Dihydroetorphines and their preparation

ABSTRACT

The present invention provides a process for the preparation of a compound of formula (VI), or a salt or derivative thereof, 
                         
wherein R 1  and R 2  are independently C 1-8  alkyl and * represents a stereocentre.

This invention relates to a new process for making dihydroetorphine, to(S)-dihydroetorphine per se as well as to intermediates prepared duringits synthesis.

(R)-Dihydroetorphine (shown below) is a potent analgesic drug.

It is mainly used in China in sublingual form at doses ranging from 20to 180 μg. Compared to other analgesics it is reported to cause stronganalgesia and relatively mild side effects. The use of(R)-dihydroetorphine in transdermal patches is also disclosed inJP-10-231248. As far as the applicant is aware, however, no such patchis commercially available.

Dihydroetorphine is a variant of etorphine. (R)-Etorphine is anextremely powerful opioid used for anaesthetising animals, e.g.elephants. It was developed in the 1960s and synthetic routes for itspreparation are well known. Example 12 of GB 925,723, for instance,discloses a synthesis of etorphine wherein a Grignard reagent (propylmagnesium iodide) is added to a thebaine derivative as shown below:

The results given in Example 12 state that the α-isomer is produced upontrituration of the crude reaction product with methanol and that theβ-isomer could be crystallised from the methanolic liquors when theywere diluted with water and the liquid decanted. The applicant thereforeexpected that the synthetic route described in GB 925,723 could beapplied to dihydroetorphine and that both (R) and (S) diastereomerswould result. It was found, however, that this was not the case. Ratherthe addition of propyl magnesium halide to the dihydro thebainederivative occurred with unexpectedly high stereoselectivity and only(R) diastereomer was obtained.

As far as the applicants are aware, the (S) isomer of dihydroetorphinehas never been prepared. There is therefore a need for an alternativesynthetic route that affords (S)-dihydroetorphine and especially for aprocedure that yields (S)-dihydroetophine in a high diastereomericexcess. This isomer is required to confirm the stereochemistry of theknown stereoisomers.

The applicant has now found a process that satisfies these needs.Moreover applicant has found that the (S) isomer of dihydroetorphinepossesses useful pharmacological properties and in particular analgesiceffects.

Thus viewed from one aspect the invention relates to a process for thepreparation of a compound of formula (VI), or a salt or derivativethereof,

(wherein R¹ and R² are independently C₁₋₈ alkyl and * represents astereocentre, preferably a S stereocentre) comprising hydrolysing acompound of formula (V)

wherein R¹, R² and * are as hereinbefore defined.

In a preferred process of the present invention, the compound of formula(V) is prepared by reacting a compound of formula (IV)

(wherein R¹ is as hereinbefore defined);with a compound of formula R²M(X)p, wherein R² is C₁₋₈ alkyl, M ismetal, X is halide and p is 1 or 0).

In a further preferred process of the present invention, the compound offormula (IV) is prepared by reducing a compound of formula (III)

(wherein R¹ is as hereinbefore defined);

In a yet further preferred process of the present invention, thecompound of formula (III) is prepared by reacting a compound of formula(I)

with a compound of formula (II)

(wherein R¹ is C₁₋₈ alkyl).

Thus viewed from another aspect, the present invention provides aprocess for the preparation of a compound of formula (VI), or a salt orderivative thereof,

(wherein R¹ and R² are independently C₁₋₈ alkyl and * represents astereocentre, preferably a S stereocentre) comprising:reacting a compound of formula (I)

with a compound of formula (II)

(wherein R¹ is C₁₋₈ alkyl) to give a compound of formula (III)

(wherein R¹ is as hereinbefore defined);

reducing said compound of formula (III) to produce a compound of formula(IV)

(wherein R¹ is as hereinbefore defined);reacting said compound of formula (IV) with a compound of formulaR²M(X)_(p), wherein R² is C₁₋₈ alkyl, M is metal, X is halide and p is 1or 0, to give a compound of formula (V)

(wherein R¹, R² and * are as hereinbefore defined);(iv) hydrolysing said compound of formula (V) to produce a compound offormula (VI).

Viewed from a further aspect the invention relates to a compound offormula (VI), or a salt or derivative thereof,

wherein R¹ and R² are independently C₁₋₈ alkyl and the * represents a(S) stereocentre.

Viewed from a still further aspect the invention relates to compoundsthat are intermediates in the above-described process, i.e. to compoundsof formulae (V), (IV) and (III), or where applicable to salts orderivatives thereof, as shown below.

wherein R¹ and R² are independently C₁₋₈ alkyl and the * represents a(S) or (R) stereocentre, preferably a (S) stereocentre.

wherein R¹ is C₁₋₈ alkyl.

wherein R¹ is C₁₋₈ alkyl.

Viewed from a still further aspect the invention relates to a processfor preparing a compound of formula (III) comprising reacting a compoundof formula (I)

with a compound of formula (II)

(wherein R¹ is C₁ alkyl).

Viewed from another aspect the invention relates to compositions,preferably pharmaceutical compositions, comprising a novel compound ashereinbefore described.

Viewed from another aspect the invention relates to compounds ashereinbefore described for use in medicine (e.g. as an analgesic).

Viewed from yet another aspect the invention relates to use of acompound as hereinbefore described for the manufacture of a medicamentfor the treatment of pain.

As used herein the term “alkyl” is used to refer to a straight chained,cyclic or branched, saturated, aliphatic hydrocarbon. Preferred alkylgroups present in the compounds (II)-(VI) are straight chained alkylgroups. Preferred alkyl groups are of the formula C_(n)H_(2n+1) whereinn is 1 to 8. Typical alkyl groups include methyl, ethyl, propyl, butyl,pentyl, hexyl, heptyl and octyl. Preferred alkyl groups in the compounds(II)-(VI) are unsubstituted.

The compound of formula (I) is thebaine and is commercially available,e.g. from Tasmanian Alkaloids, Pty. Alternatively the compound offormula (I) can be prepared according to procedures described in theliterature.

In a preferred process of the invention R¹ in the compound of formula(II) is preferably C₂₋₇ alkyl, more preferably C₃₋₅ alkyl, especially C₃alkyl (e.g. n-propyl). A particularly preferred compound of formula (II)is hexen-3-one. It is commercially available, e.g. from Sigma-Aldrich.

The compound of formula (I) is reacted with a compound of formula (II)so as to produce a compound of formula (III). The reaction that thesecompounds undergo is typically referred to as a Diels-Alder reaction.The Diels-Alder reaction may be carried out under conventionalconditions known in the art. The reaction of compounds of formulae (I)and (II) may, for instance, be carried out in any conventional solvent.Solvents having boiling points in excess of 60° C. are preferred (e.g.methanol and ethanol). Ethanol is a particularly preferred solvent.

In a typical reaction between compounds of formulae (I) and (II) thecompounds are heated to reflux in excess solvent, e.g. for 10-24 hours.The process of the reaction may be monitored using, e.g. TLC and/or ¹HNMR. In a preferred reaction 1.2-15 molar equivalents, more preferably1.5-10 molar equivalents or 2-8 molar equivalents of the compound offormula (II) is used relative to the compound of formula (I). In aparticularly preferred reaction about 1.2-2 molar equivalents, morepreferably 1.3-1.8 molar equivalents, e.g. about 1.5 molar equivalentsof the compound of formula (II) is used relative to the compound offormula (I).

The reaction mixture is then cooled and concentrated. The resultingproduct, a compound of formula (III), may be obtained by a conventionalwork up procedure and optionally purified. Purification may, forexample, by carried out by crystallisation from methanol or isopropylalcohol. More preferably the compound of formula (III) crystallisesdirectly from the reaction solvent. It may optionally be recrystallised.The yield of the reaction is preferably at least 60%, more preferably atleast 65%, e.g. at least 80%. The maximum yield is 100%. The purity ofthe compound of formula (III) is preferably at least 95%, morepreferably at least 97%, still more preferably at least 99%, e.g. 99.5%.The maximum purity is 100%. Purity is preferably determined using HPLC.

In a preferred process of the invention the compound of formula (III) isof formula:

wherein R¹ is as hereinbefore defined, e.g. R¹ is C₂₋₇ alkyl, morepreferably C₃₋₅ alkyl, especially C₃ alkyl (e.g. n-propyl).

The compound of formula (III) may be reduced by any suitable knownreduction reaction but is preferably reduced using an hydrogenationreaction (e.g. using H₂ in a Parr vessel or hydrogen transfer). Thecompound of formula (III) may, for example, be hydrogenated in solvent(e.g. ethanol) with catalyst (e.g. palladium on carbon) under a pressureof hydrogen (e.g. up to 50 psi H₂). The volume of the reaction ispreferably in the range 5-80 L, more preferably 10-20 L, e.g. about 12L. The amount of catalyst used is preferably in the range 10-60% wt,more preferably 30-55% wt, e.g. about 50% wt. The reaction may becarried out at a temperature of 30-100° C., preferably at a temperatureof 40-60° C., e.g. at 50° C. or 65° C.

At the end of the reaction, any catalyst (e.g. palladium) used thereinmay be removed by filtration. The product, a compound of formula (IV),may then be isolated by a conventional work up procedure. The compoundof formula (IV) is optionally purified. For instance, washing with aC₁₋₅ alkane such as heptane removes ethanol. An advantage of thehydrogenation reaction is, however, that the compound of formula (IV)can be used without purification by chromatography and/orcrystallisation. The yield of the reaction is preferably at least 50%,more preferably at least 65%, still more preferably 85%, still morepreferably at least 90%. The maximum yield is 100%. The compound offormula (IV) is preferably obtained with a purity of at least 95%, morepreferably at least 99%, e.g. at least 99.5%. The maximum purity is100%. Purity is preferably determined using HPLC.

In a preferred process of the invention the compound of formula (IV) isof formula:

wherein R¹ is as hereinbefore defined, e.g. R¹ is C₂₋₇ alkyl, morepreferably C₃₋₅ alkyl, especially C₃ alkyl (e.g. n-propyl)

The compound of formula (IV) is reacted with a compound of formulaR²M(X)_(p) wherein R² is C₁₋₈ alkyl, M is metal (e.g. an alkali oralkaline earth metal), X is halide and p is 1 or 0, to produce acompound of formula (V). In preferred compounds of formula R²M(X)_(p),R² is C₁₋₃ alkyl, more preferably C₁₋₂ alkyl, e.g. methyl.

In further preferred compounds of the formula R²M(X)_(p) M is magnesiumor lithium, preferably magnesium. When M is Mg, p is preferably 1. WhenM is lithium, p is preferably 0. When present X is preferably Cl, Br orI. Methyl magnesium halide, especially methyl magnesium bromide andmethyl magnesium iodide, is a preferred compound of formula R²M(X)_(p),especially methyl magnesium bromide.

The reaction of the compound of formula (IV) with a compound of formulaR²M(X)_(p) is typically referred to as a nucleophilic addition reaction.When M is Mg and X is halide, the reaction is often referred to as aGrignard addition. The addition reaction may be carried out in anyconventional solvent. Preferred solvents are non-aqueous (e.g. anhydroussolvents). An example of a preferred solvent is an ether, e.g. MTBE, THFor diethyl ether. MTBE or diethyl ether are preferred. Diethyl ether isa particularly preferred solvent. THF is particularly preferred when acompound of formula R²M(X)_(p), wherein M is Mg, X is Cl and p is 2, isused.

The addition reaction is preferably carried out at a temperature in therange 20 to 60° C., more preferably 30 to 45° C., e.g. about 35° C. Anexcess of the compound of formula R²M(X)_(p) is preferably used. Inparticular 1.2-4 equivalents, more preferably 1.5-3 equivalents of acompound of formula R²M(X)_(p) is preferably used relative to thecompound of formula (IV).

The compound of formula (V) may be isolated using conventionaltechniques. It may optionally be triturated, e.g. with methanol.Additionally, or alternatively, the compound of formula (V) may bepurified by column chromatography. The compound of formula (V) may alsobe crystallised. Preferably the compound of formula (V) is crystallisedwith methanol. The yield of the reaction is preferably at least 20%,more preferably at least 30%, e.g. 20-60%, still more preferably atleast 65%. The maximum yield is 100%. The purity of the compound offormula (V) is preferably at least 95%, still more preferably at least99%, e.g. at least 99.5%. The maximum purity is 100%. Purity ispreferably determined using HPLC.

The addition reaction generates a new stereocentre in the compound offormula (V) at carbon 19. The configuration of this stereocentredepends, at least partially, on the nature of R¹ and R². Thus both (R)and (S) stereocentres may be generated. The process of the presentinvention may therefore provide a racemic mixture of compounds offormula (V). Correspondingly the present invention provides a racemicmixture of compounds of formula (VI), e.g. 19-(R) and(S)-dihydroetorphine.

In preferred processes of the invention, a (S) stereocentre is generatedat carbon 19. In particularly preferred processes, a (S) stereocentre isgenerated at carbon 19 in a diastereomeric excess of at least 85%, morepreferably at least 90%, e.g. at least 95% or at least 99%. Thus in apreferred process a compound of formula (V) is provided in the absenceof, or substantial absence of, (R)-isomer. Preferably the compound offormula (V) is provided with less than 1% wt, still more preferably lessthan 0.5% wt of (R)-isomer.

In a particularly preferred process of the invention, R¹ is C₃₋₆ alkyl(e.g. propyl), R² is C₁₋₂ alkyl (e.g. methyl) and a (S) stereocentre isgenerated in the addition reaction at carbon 19 in a diastereomericexcess of at least 85%, more preferably at least 90%, e.g. at least 95%or at least 99%.

Thus in a preferred process of the invention the compound of formula (V)is of the formula:

wherein R¹ and R² are as hereinbefore described, e.g. R¹ is C₂₋₇ alkyl,more preferably C₃₋₅ alkyl, especially C₃ alkyl (e.g. n-propyl), R² isC₁₋₃ alkyl, more preferably C₁₋₂ alkyl, e.g. methyl and the (*)represents a stereocentre, preferably a S stereocentre.

In a particularly preferred process of the invention the compound offormula (V) is:

As mentioned above, the compound of formula (V) may optionally becrystallised. In a preferred process of the invention, the compound offormula (V) is crystallised. Any conventional solvent may be used forthe crystallisation process, e.g. C₁₋₄ alcohols, water, acetone,acetonitrile, DCM and MTBE. Methanol, ethanol, water and mixturesthereof are preferred crystallisation solvents, especially ethanol/waterand ethanol. In a typical crystallisation process, an amount of thecompound of formula (V) obtained from the addition reaction is dissolvedin the chosen solvent, preferably a minimum amount thereof, and thesolution is allowed to stand, e.g. for 3-4 days. Preferablycrystallisation is carried out at −5 to 5° C., e.g. 0-4° C.

The compound of formula (V) is preferably hydrolysed with an alkalimetal hydroxide to form a compound of formula (VI). A preferred alkalimetal hydroxide is KOH. An excess of alkali metal hydroxide ispreferably used in the hydrolysis reaction, e.g. an excess of 10-40equivalents relative to the compound of formula (V). The reaction may becarried out in any conventional solvent. Diethylene glycol is apreferred solvent.

The hydrolysis reaction is preferably carried out at a temperature inthe range 150-220° C., e.g. about 180-200° C. The progress of thereaction may be monitored by conventional techniques, e.g. TLC, but willtypically take 10-20 hours, e.g. 12-18 hours. After the reaction iscomplete, the compound of formula (VI) may be isolated usingconventional techniques. The compound of formula (VI) may be triturated.The yield of the reaction is preferably at least 40%, more preferably atleast 45%, still more preferably 85%, yet more preferably at least 90%.The maximum yield is 100%. The purity of the compound of formula (VI) ispreferably at least 90%, still more preferably at least 95%. The maximumpurity is 100%. Purity is preferably determined using HPLC.

The compound of formula (VI) may also be crystallized. Preferredsolvents for use in crystallisation are AcCN and MTBE. More preferablythe compound of formula (VI) is crystallised from a C₁ alcohol and/orwater, e.g. ethanol and/or ethanol/water.

In a preferred hydrolysis reaction the stereochemistry of each of thestereocentres present in the compound of formula (V) is retained.Preferably the compound of formula (VI), e.g. 19-S-dihydroetorphine, isprovided in the absence of or substantial absence of the (R)-isomer.Preferably less than 1% wt, more preferably less than 0.5% wt, stillmore preferably less than 0.01% wt (R)-isomer is present.

Thus in a preferred process the compound of formula (VI) is:

wherein R¹ and R² are as hereinbefore described, e.g. R¹ is C₂₋₇ alkyl,more preferably C₃₋₅ alkyl, especially C₃ alkyl (e.g. n-propyl), R² isC₁₋₃ alkyl, more preferably C₁₋₂ alkyl, e.g. methyl and the (*)represents a stereocentre, preferably a S stereocentre. Preferably thecompound of formula (VI) has a purity of at least 99%, e.g. asdetermined by HPLC.

In a particularly preferred process the compound of formula (VI) is:

The compounds (V) and (VI) hereinbefore described may be converted intotheir salts and derivatives by techniques well known in the art.Preferred salts are pharmaceutically acceptable salts. Preferredderivatives are pharmaceutically acceptable derivatives. A derivativethat sometimes occurs in small amounts (e.g. <5% wt) is the 6-hydroxycompound. This is produced if the hydrolysis reaction additionallyhydrolyses the 6-methoxy group. The 6-hydroxy derivative may be isolatedby recrystallisation.

Preferred salts are those that retain the biological effectiveness andproperties of the compounds of the present invention and are formed fromsuitable non-toxic organic or inorganic acids. Adid addition salts arepreferred. Representative examples of salts include those derived frominorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodicacid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, andthose derived from organic acids such as p-toluenesulfonic acid,salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citricacid, malic acid, lactic acid, fumaric acid, trifluoro acetic acid andthe like. The modification of a compound into a salt is a technique wellknown to chemists to obtain improved physical and chemical stability,hygroscopicity, flowability and solubility of compounds.

Preferred compounds of the invention are compounds of formulae (VI),(V), (IV) and (III) as described above wherein R¹ is preferably C₂₋₇alkyl, more preferably C₃₋₅ alkyl, especially C₃ alkyl (e.g. n-propyl).In preferred compounds of formulae (VI) and (V), R² is C₁₋₃ alkyl, morepreferably C₁₋₂ alkyl, e.g. methyl. In compounds (VI) and (V) of theinvention, the stereocentre at carbon 19 is (S).

A preferred compound of formula (VI) is a compound of formula:

wherein R¹ and R² are as hereinbefore described (e.g. R¹ is C₂₋₇ alkyl,more preferably C₃₋₅ alkyl, especially C₃ alkyl (e.g. n-propyl), R² isC₁₋₃ alkyl, more preferably C₁₋₂ alkyl, and the (*) represents a (S)stereocentre.

A particularly preferred compound of formula (VI) is a compound offormula:

Further preferred compounds of the invention are those that areintermediates in the preparation of compounds of formula (VI). Thusother preferred compounds of the invention are compounds of formula(V-S):

wherein R¹ and R² are as hereinbefore described keg. R¹ is C₂₋₇ alkyl,more preferably C₃₋₅ alkyl, especially C₃ alkyl (e.g. n-propyl), R² isC₁₋₃ alkyl, more preferably C₁₋₂ alkyl), and the (*) represents a (S) or(R) stereocentre, preferably 3 (S) stereocentre.

A particularly preferred compound of formula (V) is:

Further preferred intermediates are compounds of the formulae (IVa) and(IIIa) as shown below:

As described above, the compounds of formula (III), such as (IIIa)above, may be formed by a Diels-Alder reaction with a compound offormula (II). This reaction forms a further aspect of the invention.Preferences for R¹ are as hereinbefore described.

The compounds of the present invention have various uses. The compounds(VI-S) can, for example, be used to confirm the (R) chirality of theknown dihydroetorphine products. The use of the compounds of theinvention in this way is illustrated in the examples that followhereinafter. The compounds (III) and (IV) of the invention are alsouseful in the preparation of (R)-dihydroetorphine, which is known tohave useful pharmaceutical properties.

Moreover the compounds of formulae (VI-S), (V-S), (V-R), (IV) and (III),especially compounds of formula (VI-S), may be incorporated intocompositions, preferably pharmaceutical compositions. Thus, the presentinvention also includes pharmaceutical compositions comprising acompound of the invention as hereinbefore described (e.g. compounds offormulae (VI-S), (V-S), (V-R), (IV) and (III), especially (VI-S)) andone or more pharmaceutically acceptable carriers. The compounds of theinvention, e.g. compounds of formula (VI-S) can be present alone or incombination with another active ingredient in a composition.

The compositions, e.g. pharmaceutical compositions, of the invention maytake any conventional form. Preferably, however, the compositions of theinvention are prepared in a dosage form suitable for transdermaladministration. Alternative preferred compositions of the invention areprepared in a dosage form suitable for parenteral, e.g. intravenous,administration.

By “transdermal” delivery is meant administration of the compoundshereinbefore described to the skin surface of an individual so that theagent passes through the skin tissue and into the individual's bloodstream. The term “transdermal” is intended to include transmucosaladministration, i.e., administration of the compound to the mucosal(e.g., sublingual, buccal, vaginal, rectal) surface of an individual sothat it passes through the mucosal tissue and into the individual'sblood stream.

Transdermal dosage forms of the invention include, but are not limitedto, mouth pastilles, sprays, aerosols, creams, lotions, ointments, gels,solutions, emulsions, suspensions, or other forms known to one of skillin the art. Dosage forms suitable for treating mucosal tissues withinthe oral cavity can be formulated as mouthwashes or as oral gels.Further, transdermal dosage forms include “reservoir type” or “matrixtype” patches, which can be applied to the skin and worn for a specificperiod of time to permit the penetration of a desired amount of activeingredients.

Suitable excipients (e.g. carriers and diluents) and other materialsthat can be used to provide transdermal dosage forms encompassed by thisinvention are well known to those skilled in the pharmaceutical arts,and depend on the particular tissue to which a given pharmaceuticalcomposition or dosage form will be applied. With that fact in mind,typical excipients include, but are not limited to, water, acetone,ethanol, ethylene glycol, propylene glycol, butane-1,3-dial, isopropylmyristate, isopropyl palmitate, mineral oil, and mixtures thereof toform lotions, tinctures, creams, emulsions, gels or ointments, which arenon-toxic and pharmaceutically acceptable. Moisturizers or humectantscan also be added to pharmaceutical compositions and dosage forms ifdesired. Examples of such additional ingredients are well known in theart.

Depending on the specific tissue to be treated, additional componentsmay be used prior to, in conjunction with, or subsequent to treatmentwith the compounds of the invention. For example, penetration enhancerscan be used to assist in delivering the compound to the tissue. Suitablepenetration enhancers include, but are not limited to: acetone; variousalcohols such as ethanol, oleyl, and tetrahydrofuryl; alkyl sulfoxidessuch as dimethyl sulfoxide; dimethyl acetamide; dimethyl formamide;polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; Kollidongrades (Povidone, Polyvidone); urea; and various water-soluble orinsoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60(sorbitan monostearate).

The pH of a pharmaceutical composition or dosage form, or of the tissueto which the pharmaceutical composition or dosage form is applied, mayalso be adjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

Oral gels for sublingual administration of the compounds of theinvention (e.g. compounds of formulae (VI-S)) can be prepared by mixingthe compound with one or more suitable excipients including flavouringagents. Suppositories for rectal administration of the compounds of theinvention (e.g. compounds of formulae (VI-S)) can be prepared by mixingthe compound with a suitable excipient such as cocoa butter, salicylatesand polyethylene glycols. Formulations for vaginal administration can bein the form of a pessary, tampon, cream, gel, paste, foam, or sprayformula containing, in addition to the active ingredient, such suitablecarriers as are known in the art.

For topical administration the pharmaceutical composition comprising thecompounds of the invention can be in the form of creams, ointments,liniments, lotions, emulsions, suspensions, gels, solutions, pastes,powders, sprays, and drops suitable for administration to the skin, eye,ear or nose. Topical administration may also involve transdermaladministration via means such as transdermal patches. Delivery in thisform is particularly preferred.

By intravenous administration is meant administration of the compoundshereinbefore described in the form of a liquid directly into a vein.Dosage forms suitable for intravenous administration include, but arenot limited to, solutions, emulsions and suspensions.

Thus viewed from a further aspect, the invention provides a compound ashereinbefore defined, and especially compounds of formula (VI-S), foruse as an analgesic, wherein said compound is administeredintravenously.

Typically, compositions for intravenous administration comprise sterileisotonic aqueous buffer. Where necessary, the compositions can alsoinclude a solubilizing agent. The ingredients may be supplied eitherseparately or mixed together in unit dosage form. For example theingredients may be supplied separately as a dry lyophilized powder orwater free concentrate in a hermetically sealed container, e.g. anampule or sachette indicating the quantity of active agent, and as anampoule of sterile water or buffer for mixing prior to administration.Alternatively the composition may be supplied in a pre-mixed form.

The compounds of the invention (e.g. compounds of formulae (VI-S)) maybe used in medicine, e.g. to provide analgesia. The doses of compoundsrequired will be dependent, for example, on the subject to be treated,the severity of the pain to be treated, the compound used, the mode ofadministration etc but will be readily determined by those skilled inthe art.

Thus viewed from a further aspect the invention provides a method oftreating a subject (e.g. mammal) in need of pain relief comprisingadministering to said subject a therapeutically effective amount of acompound as hereinbefore described (e.g. a compound of formula (VI-S)).It has also surprisingly been found that in standard tests for nauseaand vomiting in ferrets, neither R-DHE nor S-DHE induced nausea orvomiting in similar dose ranges as used in the tests described below.

The compounds of the invention are particularly useful in the treatmentof nociceptive and neuropathic pain.

The invention will now be described with reference to the followingnon-limiting Examples and Figures wherein:

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A shows the chemical structure and the 1H NMR spectrum for (R)-19Propyldihydrothevinol.

FIG. 1B shows the 1H NMR spectrum for (S)-19 Propyldihydrothevinol.

FIG. 2 shows the chemical structure and the X-ray structure of (R)-19Propyldihydrothevinol depicting (R)-configuration at carbon 19 and(R)-configuration of the methyl ether.

FIG. 3 shows the chemical structure and the X-ray structure of (R)-19Propyldihydrothevinol depicting the hydrogen atoms at the 7 position andat the 5 position on the same face giving the configuration as (R).

FIG. 4 shows the chemical structure and the X-ray structure of (S)-19Propyldihydrothevinol depicting the (S) configuration at carbon 19 and(R)-configuration of the methyl ether.

FIG. 5 shows the chemical structure and the X-ray structure of (S)-19Propyldihydrothevinol depicting the configuration of the 7-carbonhydrogen as (R).

FIGS. 6 and 7 show the X-ray structure of (R)-Dihydroetorphine depictingthe (R)-configuration at carbon 19.

FIGS. 8 and 9 show the X-ray structure of (S)-Dihydroetorphine depictingthe (S)-configuration at carbon 19.

FIG. 10 shows the stereochemistry of all of the chiral carbons presentin (R)- and (S)-Dihydroetorphine.

FIGS. 11 to 13 show time-course curves following intraveneousadministration of (R)- or (S)-DHE or a reference or comparatorsubstance.

FIGS. 14 to 17 show dose response curves following intravenousadministration of (R)-DHE, (S)-DHE or a reference or comparatorsubstance.

FIGS. 18 to 23 show the effects of intravenous administration of (R)- or(S)-DHE or a reference or comparator substance in the spinal nerveligation model of neuropathic pain.

EXAMPLES Preparation of (S)-Dihydroetorphine

Stage 1—Diels-Alder Reaction

Method

Thebaine was treated with hexen-3-one in a solvent as specified in thetable below and heated to reflux. After an appropriate amount of time(overnight), the reaction was cooled and the mixture evaporated. Theresulting oil was dissolved in Isopropylacetate (IPAc) and washed with1M hydrochloric acid solution. The acidic layers were combined andwashed with IPAc then basified with sodium bicarbonate solution andfinally extracted into dichloromethane (DCM). The DCM layer wasevaporated to give a yellow solid.

TABLE 1 Summary of Experiments, Stage 1 Temp Scale Conditions (° C.)Comments 500 mg Benzene (20 vols) Reflux Overnight reflux Hexen-3-one(2.0 mol eq) gave approx 40% completion 4.5 g Methanol (10 vols) Reflux68% completion Hexen-3-one (2.0 mol eq) by NMR 4.5 g Ethanol (10 vols)Reflux >95% completion Hexen-3-one (7.5 mol eq) by NMR. 70% isolatedyield

Using ethanol as the solvent, the final isolated yield of product was70% as a light yellow solid, after work-up and the quality by ¹H NMRlooked very good.

Stage 2—Hydrogenation

Method

The 19-propylthevinone (4.1 g) intermediate from stage 1 washydrogenated in ethanol (60 ml) using palladium on carbon (1 g; 10%)under a pressure of hydrogen up to 50 psi. The temperature of the vesselwas maintained at ˜50° C. and the pressure maintained at 50 psi until nofurther uptake of hydrogen was noted. The catalyst was filtered and thesolvent removed by distillation under vacuum. Isolated yield was 91% intotal, giving a 3.8 g of product

TABLE 2 Summary of Experiments, Stage 2 Scale Conditions Temp (° C.)Comments 4.1 g Ethanol (60 ml) 50° C. −91% isolated yield 1 g Pd/C (10%)hydrogen (50 psi)

Stage 3—Grignard Addition

Method

19-propyldihydrothevinone (Stage 2 product) was dissolved in diethylether (35 vols). Methyl magnesium bromide (92.6 mol. eq.) was added tothis solution over 5 minutes at 20-25° C. (small exotherm). Theresulting mixture was then heated to ˜40° C. internal temperature for ˜2hours, before cooling and quenching with ammonium chloride solution. Themixture was extracted with 2-methyl THF and the organic layersevaporated in vacuo to give a viscous oil.

TABLE 3 Summary of Experiments, Stage 3 Scale Conditions Temp (° C.)Comments 0.13 g 2-diethyl ether (25 vols) 40° C. Good quality material3M MeMgBr (1.5 eq) produced 0.79 g 2-diethyl ether (25 vols) 35° C.Stirred overnight. 93% 3M MeMgBr (1.5 eq) purity after work-up. Crudeproduct triturated in methanol to give 0.32 g pure material and 0.5 gimpure residues.  2.2 g 2-diethyl ether (35 vols) 35° C. 2.6 g crude(~90% pure) 3M MeMgBr (2.6 eq) isolated. Triturated in methanol to give1.6 g of pure material.

The sole product of the Grignard addition is the (S)-enantiomer. No(R)-enantiomer was detected.

Stage 4—Crystallisation of (R) and (S)-19-propyldihydrothevinol

In order to prepare a single crystal of high quality for x-raycrystallography, a series of experiments were run in many solvents todetermine the best solvent system for growing a single crystal of19-propyldihydrothevinol. The experiments are summarized in table 4below. The R-enantiomer was prepared using an alternative method.

In general the crystallisation method used was as follows: a smallamount of solid 19-propyldihydrothevinol (obtained from Stage 3) wasdissolved in just over the minimum amount of solvent. The solution wasallowed to stand for up to 3-4 days and the solvent removed byfiltration or decanting in order to isolate single crystals.

TABLE 4 Summary of Re-crystallisations Co- Diastereo- Temper- Solventsolvent isomer ature Crystals Comments MTBE None (S) RT Yes Highquality- submitted for X-ray AcCN None (S) RT Yes High quality DCM None(R) Dissolved Yes High quality- Hot submitted for X-ray Acetone None (R)Dissolved Yes High quality- Hot submitted for X-ray Ethanol None (R)Dissolved Yes High quality- Hot submitted for X-ray

The ¹H NMR spectra for each of the diastereomers are shown in FIG. 1Aand FIG. 1B.

Stage 5—Hydrolysis of (S)-19-Propyldihydrothevinol

Method

The (S)-19-propyldihydrothevinol (from Stage 3) was dissolved indiethylene glycol (17 vols) and treated with potassium hydroxide (˜20eqs) and heated to ˜195° C. for 12-18 hours. After this time, thereaction mixture was cooled to room temperature and quenched into water(40 vols). The resulting solution was acidified to pH 9-10 using solidammonium chloride and the mixture extracted with DCM (3×50 vols). Thecombined organic extracts were evaporated in vacuo to a crude oil(approx. 40% purity). The purity was increased with repeatedtriturations in methanol until a yellow solid was formed and isolated ingood purity (>95%).

The product was recrystallised from several solvents and crystals wereobtained from acetonitrile. These were used for X-ray crystallographicstudies.

R-enantiomer was obtained using an analogous reaction.

X-Ray Crystallography Studies

All X-ray crystallography experiments were carried out on an OxfordXcalibur single crystal diffractometer or a Nonius Kappa diffractometer.Both machines were using Molybdenum K alpha X-ray sources and CCDdetectors.

(R) and (S) 19-Propyldihydrothevinol

Several batches of both (R) and (S) 19-propyldihydrothevinol weresubmitted for X-ray crystallography.

The X-ray structures are shown in FIGS. 2-5.

FIGS. 2 and 3 show the X-ray structure of (R)-19 Propyldihydrothevinol.From the X-ray it can be clearly seen that it has the (R)-configurationat carbon 19. This can be assigned with respect to chiral methyl ether,which retains the (R)-configuration from the thebaine starting material.

Additionally from FIG. 3, where the hydrogen atoms are showing, it canbe seen that the hydrogen at the 7 position is on the same face as thehydrogen at position 5 (next to the furan ring), giving theconfiguration as (R).

In this way all of the chiral carbons have now been assigned and aredepicted in FIG. 10.

TABLE 6 Other Information (R)-19-Propyldihydrothevinol Property ValueSymmetry cell setting Monoclinic Symmetry space group name H-M P2(1)Loop symmetry equiv pos as xyz ‘x, y, z’ ‘−x, y + ½, −z’ Cell length a 11.0464(6) Cell length b  12.4554(7) Cell length c  16.2271(7) Cellangle alpha  90.00 Cell angle beta  98.481(5) Cell angle gamma  90.00Cell volume 2208.2(2)

FIGS. 4 and 5 show the X-ray structure of (S)-19-Propyldihydrothevinol.

From FIG. 4 it can be clearly seen that it has the oppositestereochemistry of (S)-configuration at carbon 19 to the crystal shownin FIG. 3. This can be assigned with the respect to chiral methyl ether,which retains the (R)-configuration from thebaine starting material.

Again the 7-carbon hydrogen (show in FIG. 5) shows that theconfiguration at this carbon is also (R), as with the firstdiastereoisomer.

Therefore we can now safely conclude that the only difference betweenthe compounds by X-ray crystallography is the stereoconfiguration atcarbon 19.

TABLE 7 Other Information (S) 19-Propyldihydrothevinol Property ValueSymmetry cell setting Monoclinic Symmetry space group name H-M P2(1)Loop symmetry equiv pos as xyz ‘x, y, z’ ‘−x, y + ½, −z’ Cell length a 13.8650(3) Cell length b  10.8560(2) Cell length c  15.4030(4) Cellangle alpha  90.00 Cell angle beta  99.7500(8) Cell angle gamma  90.00Cell volume 2284.95(9)

(R) and (S)-Dihydroetorphine

The X-ray structures are shown in FIGS. 6-9.

FIGS. 6 and 7 shows the X-ray structure of (R)-dihydroetorphine. It canclearly be seen from these Figures that it has the (R) configuration atcarbon 19. This can be assigned with respect to the chiral methyl ether,which retains the (R)-configuration from the original starting material,thebaine.

TABLE 8 Other Information (R)-Dihydroetorphine Property Value Symmetryspace group name P 2₁ 2₁ 2 Loop symmetry equiv pos as xyz ‘x, y, z’ ‘,‘−x, ½ + y, −z’ Cell length a  16.3405(7) Cell length b  35.605(2) Celllength c   7.5209(3) Cell angle alpha  90.00 Cell angle beta  90.00 Cellangle gamma  90.00 Cell volume 4375.69

FIGS. 8 and 9 show the X-ray structure of (S)-dihydroetorphine. It canclearly be seen from these Figures that it has the (S) configuration atcarbon 19. This can be assigned with respect to the chiral methyl ether,which retains the (R)-configuration from the original starting material,thebaine.

Additionally the 7-carbon hydrogen (shown in FIG. 9) shows that theconfiguration at this carbon is also (R), as with the firstdiastereoisomer.

Therefore it can be concluded that the only difference between the 2compounds by X-ray crystallography is the stereoconfiguration at carbon19.

TABLE 9 Other Information (S)-Dihydroetorphine Property Value Symmetryspace group name H-M P2(1) Loop symmetry equiv pos as xyz ‘x, y, z’ ‘,Rotation axis (2 fold): ‘−x, −y, z’ Screw axis (2 fold): ‘½ − x, ½ + y,−z’ Screw axis (2 fold): ‘½ + x, ½− y, −z’ Cell length a   7.2310(3)Cell length b  14.0795(6) Cell length c  10.6984(5) Cell angle alpha 90.00 Cell angle beta  96.226(4) Cell angle gamma  90.00 Cell volume1082.77

Optimisation of Process

The following methods and equipment were used:

Method 38XB and UFC-LC-MUN-1 area reverse phase, gradient HPLCprocedures using an Xbridge C18 column and a mobile phase consisting ofacetonitrile and 0.01M ammonium acetate pH 9.2.

NMR was carried out using a Bruker Avarice 400 MHz spectrometer

MS was carried out using a ZMD Micromass mass spectrometer

LC was carried out using an Agilent 1100 HPLC system

Stage 1: Diels-Alder Reaction

The initial method employed was as described above. The main contaminantwas identified as the 7β-isomer shown below.

It was subsequently discovered that both the purity and recovery of20-α-ethylthevinone could be improved by reducing the 1-hexen-3-onecharge from 2.8 equivalents to 1.8 equivalents, Table 10. A furtherimprovement was achieved by using 1.5 equivalents of 1-hexen-3-one(added in 2 portions of 1.4 eq and 0.1 eq) and upon completion of thereaction, removing 0.5 volumes of solvent via distillation. Upon coolingthe resulting solution the product precipitated as a solid (aged for 1hr) and was filtered.

Procedure: To a 1 L (3-neck) flask fitted with an overhead stirrer andreflux condenser the following were charged, thebaine (0.32M, 100 g, 1eq), EtOH (250 mL) and 1-hexen-3-one (90%, 0.45M, 58 mL, 1.4 eq). Themixture was heated at reflux for 13 hrs and analysed by ¹H NMR and foundto contain starting material (˜4.5 molar %). An additional 0.1 eq of1-hexene-3-one was added and the mixture heated for a further 2 hrs,before stirring overnight at room temperature. Analysis showed startingmaterial (˜2.8 molar %). The material was transferred to a roundbottomed flask (500 mL) (flask washed with EtOH 20 mL). EtOH (˜65 mL)was removed in vacuo at 50° C. and the resulting precipitated solidstirred at 5° C. for 1 hr before filtering. The solid was washed withice-cold EtOH (4×20 mL) and pulled dry on the filter for ˜1.5 hrs. Whitesolid (105.4 g, 80%).

TABLE 10 Scale Conditions Temp (° C.) Comments  10 g 1 eq thebaineReflux Yield 69%, 9.03 g, contains 6% 2.8 eq 1-hexen-3-one (bath temp of7β-isomer - 92% purity 2.5 vol EtOH 94° C.)  2 g 1 eq thebaine RefluxYield 55%, 1.44 g. Material 1.8 eq 1-hexen-3-one (bath temp precipitatedon cooling - 2.5 vol EtOH 94° C.) >99.5% purity 100 g 1 eq thebaineReflux Yield 80%, 105.4 g. Material 1.5 eq 1-hexen-3-one (bath tempprecipitated. 2.5 vol EtOH 101° C.) >99.5% purity

Analytical Methods and In-Process-Checks (IPCs)

¹H NMR (400 MHz) was used for IPCs plus HPLC method 38XB during labwork.

Reaction was deemed complete when <5 molar % starting material remainedby ¹H NMR based on the signals at δ 5.05 ppm and 5.3 ppm (CDCl₃)

For confirming purity and for LC-MS work method UFC-LC-MUN-1 was used inthe analytical lab.

TLC (5% MeOH/95% DCM) Iodoplatinate stain: R_(f)=0.25 Thebaine,R_(f)=0.66 (7α)-20-ethylthevinone.

Analytical Summary

HPLC (% a/a) Appearance 7α 20-ethylthevinone Thebaine 7β20-ethylthevinone RT/RRT 10.69/1.0 5.82/0.54 11.67/1.09 Whitesolid >99.5 none None

¹H NMR (CDCl₃, 400 MHz); δ=0.80 (3H, t), 1.4 (1H, m), 1.6 (3H, sext.),1.9 (1H, d), 2.0 (1H, br), 2.4-2.6 (8H, m), 2.9 (2H, br), 3.35 (2H, d),3.6 (3H, s), 3.85 (3H, s), 4.6 (1H, s), 5.6 (1H, d), 6.0 (1H, d), 6.55(1H, d), 6.7 (1H, d)

¹³C NMR (CDCl₃, 75 MHz); δ=13.71, 16.89, 22.49, 30.25, 43.26, 43.51,45.57, 45.72, 47.40, 49.94, 53.78, 56.68, 60.06, 81.52, 95.84, 113.61,119.36, 125.89, 134.07, 135.53, 141.87, 148.07

MS; [M+H]⁺=410.3

LC; >99.5% purity

TLC; 5:95; MeOH:DCM; single spot rf=0.66

Advantages of Optimised Process

Increased yield to 80%

Amount of 1-hexen-3-one has been reduced to 1.5 equivalents with nodecrease in conversion or yield.

Decrease in equivalents of 1-hexen-3-one allows for an improvedisolation (direct crystallisation from reaction solvent), which givesmaterial of very high purity.

Volume efficiency is very high (maximum ˜4 volumes total).

Purity improved to >99% (by HPLC).

IPC shows completion at >97% conversion by ¹H NMR (<3% thebaine).

Stage 2 Hydrogenation

The results of the development work for the hydrogenation stage areoutlined in Table 11 below. The reaction has been ‘stressed’ both interms of catalyst loading and reaction temperature. In addition, byreducing the reaction volume from 17 vol to 12 vol, the quality of theproduct and isolation of the product has been improved.

Interestingly both the starting material and product are thermallystable to ˜80° C. over 1-2 hours which allows for higher reactiontemperature, increasing solubility of both the starting material andproduct, during reaction. It was found that in this case the solubilitywas key to good reactivity and higher temperatures were employed duringscale-up to achieve completion of reaction.

In the final scale-up reaction the temperature increased out of “normal”range during the initial heating of the reaction vessel and a fastreaction was observed (hydrogen uptake). On reducing the temperature to55° C. the reaction decreased significantly and only on further additionof catalyst and an increase in temperature to ˜65° C. did the reactionachieve completion.

Isolation of the product was simplified by warming the reaction mixtureto 77° C., allowing sub-reflux temperature to occur and then filteringthe catalyst from the reaction mixture. The resulting solution wasinitially reduced in volume by distillation, however it was found thatthe solution could be cooled in an ice bath and high purity material wasisolated by filtration of the crystallised solid in good yield (72%).

Procedure: 20-Ethylthevinone (0.244M, 100 g) was charged to a 2 L Parrhydrogenation vessel. 10% Pd/C (50% wet, 10 g) was slurried in EtOH (200mL) and charged to the hydrogenation vessel. EtOH (10 was charged to thevessel, the vessel was sealed and inerted with argon (×4). The vesselwas refilled with hydrogen to 50 psi (×2) and finally left at 50 psi.The temperature was set to 35° C. The internal temperature peaked at 82°C. and was allowed to cool back to room temperature overnight (potexotherm). The vessel was refilled with H₂, sampled and analysed by LCand found not to be complete. The vessel was heated to an internaltemperature of 55-65° C. and the reaction progress monitored by LC—thehydrogen pressure was maintained at 50 psi throughout by periodicrefills. After 24 hrs an additional catalyst charge (5 g) was made andthe reaction continued. After an additional 16 hrs the reaction wascomplete by LC and ¹H NMR. The internal temperature was raised to 68° C.and the mixture transferred under vacuum to a 3 L rbf. The Parr vesselwas flushed with hot EtOH (200 mL) and the wash transferred to the rbf.The mixture was heated to 77° C. before filtering (GF/F paper). Thecatalyst bed was washed with hot EtOH (1×300 mL) and the filtrateallowed to cool to room temperature. The filtrate was cooled in anice-water bath for 50 min before filtering. The collected solid waswashed with ice-cold EtOH (1×100 mL), heptane (1×300 mL) and pulled dryfor 1.5 hrs. White solid (72 g, 72%).

TABLE 11 Scale Conditions Temp (° C.) Comments  4.5 g 1 eq20-ethylthevinone, Pot temp After 16 hrs, 1 g sample Pd/C (50% wet, 2.71g),   55° C. (0.7% SM left) was 14 vol EtOH, 25 psi H₂ removed. Reactioncontinued until complete. Yield 90%, 3.4 g 0.680 g 1 eq20-ethylthevinone, Pot temp Yield 85%, 0.580 g Pd/C (50% wet, 0.034 g),  55° C. 70 vol EtOH, 50 psi H₂  2.32 g 1 eq 20-ethylthevinone Pot tempAfter 16 hrs reaction (547-089-1), Pd/C   55° C. complete. (50% wet,0.232 g), Reaction filtered, 17 vol EtOH, 50 psi H₂ concentrated to ~⅓vol and ppt collected. Yield 64%, 1.494 g  0.6 g 1 eq 20-ethylthevinonePot temp Reaction complete (547-090-1), Pd/C   55° C. after 16 hrs.Workup (50% wet, 0.60 g), not complete. 17 vol EtOH, 50 psi H₂   100 g 1eq 20-ethylthevinone, Maximum Product crystallised Pd/C (50% wet, 15 g),pot temp from reaction mixture 12 vol EtOH, 50 psi H₂   82° C. afterremoval of Optimal temp catalyst. Yield 72% ~60° C. purity >99%

Analytical Methods and In-Process-Checks

¹H NMR (400 MHz) was used for IPCs plus HPLC method 38XB during labwork. Reaction progress was monitored by LC analysis: the reactioncarried out on the 100 g scale showed no 20-ethylthevinone and 96% of20-ethyldihydrothevinone.

For confirming purity and for LC-MS work method product from the 100 gscale reaction was used in the analytical lab.

Analytical Results

Appearance HPLC (a/a %) Comments White solid >99 No 20-ethylthevinoneremained. No other impurity detected.

Analysis ¹H NMR (CDCl₃; 400 MHz); δ=0.75 (1H, t, t), 0.9 (1H, t), 1.35(1H, t, d), 1.5-1.75 (7H, m), 2.1 (1H, t, d), 2.3 (5H, m), 2.5 (2H, q),2.6-2.7 (3H, m), 3.0 (1H, q, t), 3.1 (1H, d), 3.5 (3H, s), 3.9 (3H, s),4.5 (1H, d), 6.6 (1H, d), 6.7 (1H, d).

¹³C NMR (COCl₃; 75 MHz); δ=13.73, 16.99, 17.31, 21.98, 28.67, 30.70,35.17, 35.66, 43.51, 45.24, 45.78, 48.28, 48.91, 52.26, 56.76, 61.35,94.96, 114.01, 119.16, 128.71, 132.47, 141.76, 146.80

LC; >99%

Residual Solvent (by ¹H NMR); No residual ethanol

Advantages of Optimised process Reducing the reaction volume from 17 volto 12 vol allows the direct crystallisation of the product.

Product isolated in 72% yield with a purity >99%.

A temperature of about 65° C. would appear to be optimum for solubilityand reactivity.

Drying the solid on the filter bed and washing with heptane removesethanol traces to an acceptable level ready for the next stage.

Stages 3 and 4: Grignard and crystallisation

The results of the work are summarised in Table 12 below. Differentethereal solvents were investigated, with diethyl ether giving the bestquality material, although the difference between diethyl ether and MTBEwas found to be relatively minor.

Generally, the crude material obtained from the Grignard reactioncontained two main impurities (˜10% of each, LC-MS). Both impuritieshave the same mass [M+H]⁺=428.4) as the product). One of the twoimpurities has been tentatively assigned as the constitutional isomer,which results from the ring-closure ring-opening reaction of excessGrignard reagent on 20-ethyldihydrothevinone.

The second impurity, which has a similar retention time (LC-MS) to theproduct is believed to be the diastereoisomer(R)-19-propyldihydrothevinol.

Both impurities are removed efficiently by the methanolre-crystallisation.

The role of the reaction temperature was investigated with diethyl etherand it was found that the reaction profile, both in terms of reactionprogress and impurities (LC analysis) were comparable at roomtemperature and at reflux temperature.

Thus, the observed differences in purity noted in the reactions arethought to arise from the different purification procedures(re-crystallisation using an oil bath and reflux condenser with magneticstirrer, or trituration via rotation on a rotary evaporator withmethanol).

Methylmagnesiumiodide was also utilised and gave comparable results tothe bromide.

Procedure: 20-Ethyldihydrothevinone (0.073M, 30 g) was dissolved (cloudysolution) in anhydrous diethyl ether (1050 mL; 35 vols). Methylmagnesiumbromide (0.189M, 63 mL) was added drop wise over 1 hr maintaining theinternal temperature below 28° C. The resulting white suspension washeated at reflux for 5 hrs, cooled to room temperature and stirred undera nitrogen atmosphere overnight. An aliquot (˜0.3 mL) was removed andquenched with sat. NH₄Cl (˜1.0 mL) and analysed by LC (upper layer fromaliquot diluted with MeCN (˜1 mL). The reaction was continued until thelevel of starting material was less than 5%. The reaction was quenchedby the addition of sat. NH₄Cl (138 mL) to the reaction mixturemaintaining the internal temperature below 30° C. The mixture was phaseseparated, the aqueous phase extracted with diethyl ether (1×200 mL) andthe combined organic phase dried (MgSO₄). The solution was concentratedin vacuo to yield a viscous oil (33.4 g). MeOH (100 mL) was added andthe mixture heated to a bath temperature of 60° C., before cooling toroom temperature. The solid was filtered, washed with ice cold MeOH(3×25 mL), washed with heptane (1×25 mL) and pulled dry. White solid (21g, 68%)

TABLE 12 Temp Scale Conditions (° C.) Comments  1.0 g 1 eq 20-EtDHT, 60°C. Crude material purified by 1.2 eq MeMgBr column chromatography toyield 6.1 vol 2-MeTHF two main fractions: 180 mg of impure product plusan unidentified material (140 mg)  0.5 g 1 eq 20-EtDHT, reflux 0.44 g ofoily gum which looks 2.6 eq MeMgBr okay by ¹H NMR. Trituration 35 volEt₂O from MeOH yielded two samples: 160 mg (96%) and 150 mg (90%)  0.6 g1 eq 20-EtDHT, reflux 0.45 g of oily gum. Purity = 77% 2.6 eq MeMgI (byLC). Trituration yielded 35 vol Et₂O 110 mg of 94.1% purity. 30.0 g 1 eq20-EtDHT reflux <2% SM and 81% product. 2.6 eq MeMgBr After work-up andre- 35 vol Et₂O crystallisation from MeOH: yield 68%, 21 g, >99% purity.  1 g 1 eq 20-EtDHT rapid. After work-up and trituration 2.6 eq MeMgBraddition, with MeOH: yield 54%, 35 vol TBME then 0.56 g, 96% purityheated 45° C./4 h   1 g 1 eq 20-EtDHT rapid After work-up andtrituration 2.6 eq MeMgBr addition, with MeOH: yield 52%, 0.54 g, 35 volCPME then 95.7% purity heated 45° C./4 h   10 g 1 eq 20-EtDHT rapidAfter completion of MeMgBr 2.6 eq MeMgBr addition, addition: 1.2%20-EtDHT, 85% 35 vol TBME then product (HPLC). After heating, heatedwork-up and trituration 35° C./4 h with MeOH: yield 75%, 7.79 g, 96.4%purity   1 g 1 eg 20-EtDHT rapid After completion of MeMgBr 2.6 eqMeMgBr addition, addition: 0.3% 20-EtDHT, 84% 35 vol Et₂O no heating,product (HPLC). After work-up stirred 4 h and trituration with MeOH:yield 58%, 0.60 g, 94.8% purity

Analytical Methods and In-Process-Checks

¹H NMR (400 MHz) was used plus HPLC method 38XB during lab work. Thereaction was monitored by LC analysis of a quenched (sat NH₄Cl) reactionaliquot: <2.0% 20-ethyldihydrothevinol and 71%(S)-19-propyldihydrothevinol.

For confirming purity and for LC-MS work method UFC-LC-MUN-1 was used inthe analytical lab.

Analytical Results

Appearance HPLC (a/a %) Comments White solid 94.1 From MeMgI Whitesolid >99 Crude material had an overall purity of 81% and contained <2%starting material

¹H NMR (CDCl₃, 400 MHz); δ=0.75 (1H, m), 0.85 (3H, t), 0.95-1.1 (6H, m),1.3 (1H, m), 1.5-1.7 (7H, m), 1.8 (1H, t), 2.0 (1H, t, d), 2.1-2.4 (6H,m), 2.6 (1H, d), 2.7 (1H, t, d), 3.0 (1H, d), 3.5 (3H, s), 3.8 (3H, s),4.3 (1H, s), 4.7 (1H, s), 6.5 (1H, d), 6.7 (1H, d)

¹³C NMR (CDCl₃, 75 MHz); δ=15.12, 16.96, 18.04, 21.91, 25.55, 29.88,31.99, 35.53, 36.05, 38.97, 43.53, 45.17, 46.19, 49.09, 50.77, 52.72,56.93, 61.32, 80.34, 97.05, 114.21, 119.06, 128.84, 132.48, 141.63,146.97

LC; >99%

Advantages of Optimised Process

Reaction works in a range of ethereal solvents, although diethyl etherappears to give the cleanest crude product.

Material of excellent purity is obtained following a re-crystallisationfrom methanol.

Stage 5: Hydrolysis

The reaction was run without significant changes (Table 13).Recrystallisation was carried out from an ethanol/water mixture thenethanol.

Procedure: (S)-DHE (10 g) was added to EtOH (60 mL) and heated at refluxuntil dissolved. Water (32 mL) forming a hazy solution which was allowedto cool to room temperature over ˜2 hrs. The white solid was collectedby filtration. (4.26 g, 42 wt % recovery). Purity 98%. Overall wt %yield=45%

TABLE 13 Temp Scale Conditions (° C.) Comments 15 g (S)-19propyldihydrothevinol (1 eg), 185 Reaction heated at KOH (20.85 eq),diethylene glycol 185° C. for ~18 hrs (16.6 vol) with air condenser.Yield of crude 107%, purity 95%

Analytical Methods and In-Process-Checks

¹H NMR (400 MHz) was used plus HPLC method 38XB during lab work forIPCs. The reaction was monitored by LC analysis and quenched when no(S)-19-propyldihydrothevinol remained. The reaction was 92% complete.

For confirming purity and for LC-MS work method UFC-LC-MUN-1 was used inthe analytical lab.

Analytical Results

Appearance HPLC (a/a %) Tan solid 95.0

¹H NMR (CDCl₃; 400 MHz); δ=0.7 (1H, m), 0.8 (3H, t), 1.0-1.1 (5H, m),1.3 (1H, m), 1.5-1.8 (6H, m), 1.85 (1H, t), 1.95 (1H, t, d), 2.1-2.3(5H, m), 2.6 (1H, d), 2.7 (1H, t), 3.0 (1H, d), 3.5 (3H, s), 4.3 (1H,s), 4.8 (1H, s), 6.0 (1H, br), 6.4 (1H, d), 6.7 (1H, d)

¹³C NMR (CDCl₃, 75 MHz); δ=15.10, 16.95, 18.00, 21.99, 25.37, 29.82,31.96, 35.43, 36.15, 38.93, 43.51, 45.22, 46.50, 49.04, 52.72, 61.33,80.42, 97.38, 116.61, 119.46, 127.92, 132.07, 137.56, 145.67

LC; 98.8%

Chiral LC 99.44% (S)-DHE. 0.554 (R)-DHE

Advantages of Optimised Process

Significant improvement in the purity of the crude material obtainedfrom the reaction to 95%_(.)

Removal of the methanol trituration—material recrystallised fromethanol/water and ethanol.

Stage 6: Recrystallisation

An increase in the water content to ˜35% (total volume 9.2 vol) resultedin the formation of a white solid. Further recrystallisations fromethanol and ethanol/water mixtures improved the purity of the material(Table 14).

Procedure:

(1) (S)-DHE (3.0 g) was added to EtOH (10 mL) and the suspension heatedat reflux producing an orange solution. The solution was allowed to coolto room temperature over 16 hrs. The resulting white solid was collectedby filtration (2.1 g, 70 wt %). Purity >99%.

(2) (S)-DHE (1.8 g) was added to EtOH (7 mL) and the mixture heated atreflux until in solution. Water (2 mL) was added and the hazy solutionallowed to cool to room temperature over 2 hrs. The resulting whitesolid was collected by filtration (1.29 g, 72 wt %).

TABLE 14 Temp Scale Conditions (° C.) Comments  10 g 9.2 vol of ~65/35reflux Material heated in EtOH, then EtOH/water water added. Whitesolid, 42 wt % recovery, 98% purity 3.0 g 3.3 vol (EtOH) reflux Whitesolid, 70 wt % recovery, >99% purity 1.8 g 4.5 vol (20% reflux Wateradded at reflux, then allowed water/80% EtOH) to cool. 72 wt % recovery,>99%

Analytical Methods and In-Process-Checks

¹H NMR (400 MHz) was used plus HPLC method 38XB during lab work forIPCs.

For confirming purity and for LC-MS work method UFC-LC-MUN-1 was used inthe analytical lab. For confirming chiral purity method UFC-LC-MUN-2 wasused in the analytical lab.

Analytical Results

Chiral % purity Appearance HPLC (a/a %) (HPLC) White solid >99% 99.98

Advantages of optimised process Recrystallisation from ethanol/water orethanol produces material of good overall purity and <0.02% (R)-DHE

Use of (R) and (S) Dihydroetorphine in the Treatment of Pain

Effects of (R) and (S)-Dihydroetorphine in the Tail Flick Test ofNociception in the Rat

The test model used is well known in the art and is described in J.Pharmacol Exp Ther, 1941, 72, 74-79 (D'Amour et al, A method fordetermining loss of pain sensation)).

The objective of this study was to assess the potential analgesiceffects of R- and S-isomers of dihydroetorphine (R-DHE and S-DHE), atdoses of 0.1, 0.3 and 0.5 μg/kg (R-DHE) and 3, 10 and 30 μg/kg (S-DHE),in a tail flick test designed to detect effects on nociception in rats.Morphine hydrochloride was used as a reference substance and fentanylcitrate was used as a comparator substance.

Test Substances and Materials

Test Substances, Reference Substance and Vehicle

Test substance: Dihydroetorphine (R-DHE colourless liquid, used as freebase) and dihydroetorphine (S-DHE liquid; used as free-base)

Vehicle for test substance: Citrate buffer (citric acidmonohydrate:sodium citrate:sodium chloride:water for irrigation, in theratio, 0.03:0.10:0.86:90.01 (g:g:g:mL)) [citric acid monohydrate (whitepowder, Sigma, UK), sodium citrate (Sigma, UK), sodium chloride (whitesolid; Merck), sterile water for irrigation (clear liquid; BaxterHealthcare, UK)]

Reference substance: Morphine hydrochloride (white powder; MacfarlanSmith, Edinburgh, UK)

Comparator substance: Fentanyl citrate (white powder; Sigma, UK)

Test Reference and Comparator Substance Storage

The test substances were stored at room temperature, protected fromlight, and the reference and comparator substances were stored at roomtemperature.

Route of Administration and Dose Levels

The route of administration of R- and S-isomer forms of DHE and thevehicle was intravenous. A possible route of administration in humans isintravenous. The doses of the R-DHE were 0.1, 0.3 and 0.5 μg/kg. Thedoses of the S-DHE were 3, 10 and 30 μg/kg.

The dose of morphine was 5 mg/kg. The route of administration ofmorphine was intravenous.

The doses of fentanyl were 0.5, 2 and 6 Mg/kg. The route ofadministration of fentanyl was intravenous.

Animals

-   Species: Rat-   Strain: Sprague-Dawley-   Sex: Male-   Number of animals: 111 animals were allocated to study; the    remaining 9 animals were returned to stock-   Age range: 9 to 11 weeks (based on the average body weight)-   Weight range: 198 to 258 g-   Acclimatisation: 6 days after delivery, before commencing the study    investigation-   Source: Harlan UK Ltd

Animal Identification and Randomisation

Each animal was arbitrarily allocated a unique identification numberwhich appeared on the data sheets and cage cards. Animals wereidentified by a waterproof tail mark.

Animal Health and Welfare

All studies were conducted in accordance with the legislation under theAnimals (Scientific Procedures) Act 1986, with UK Home Office Guidanceon the implementation of the Act and with all applicable Codes ofPractice for the care and housing of laboratory animals. The procedureadopted in this study is covered in procedure number 213N, which has amoderate severity limit.

Housing and Environment

Animals were housed in groups of up to 5 in sawdust filled solid-bottomcages. During the acclimatisation, the rooms and cages were cleaned atregular intervals to maintain hygiene. The rooms were illuminated byfluorescent lights set to give a 12 h light-dark cycle (on 07.00, off19.00), as recommended in the Home Office Animals (ScientificProcedures) Act 1986. The rooms were air-conditioned and the airtemperature and relative humidity measured. During the acclimatisationperiod room temperature was maintained (range 19° C. to 22° C.) andhumidity levels were within the range 22% to 44%. The procedure roomtemperature was maintained (range 20° C. to 21° C.) and humidity levelswere within the range 22% to 26%.

Diet and Water

A diet of RM1(E) SQC (Special Diets Services, Witham, UK) and mains tapwater were offered ad libitum. Each batch of diet was delivered with anaccompanying certificate of analysis detailing nutritional compositionand levels of specified contaminants (e.g. heavy metals, aflatoxin andinsecticides). The water was periodically analysed for impurities andcontaminants. The criteria for acceptable levels of contaminants instock diet and water supply were within the analytical specificationsestablished by the diet manufacturer and water analytical service,respectively.

Health Status

The animals were examined on arrival and prior to the study; all animalswere healthy and considered suitable for experimental use.

Experimental Design

Formulation of the Test, Reference and Comparator Substances

The citrate buffer was prepared by accurately weighing the appropriatequantities of each component and dissolving them in sterile water forirrigation. When the components were fully dissolved the osmolality andpH of the solution were measured. The vehicle was deemed acceptable asthe pH was 5.01, which was within the range pH 4.8 to 5.2 and theosmolality was 296 mOsmol/kg, between the range of 280 to 300 mOsmol/kg.The vehicle was then terminally filtered through a Millex GV stericupunder aseptic conditions and stored at 2° C. to 8° C. prior to use.

The test substances, DHE (R- and S-isomers), were formulated for dosingas solutions in citrate buffer. The desired concentrations (0.02, 0.06and 0.10 μg/mL for the R-DHE, and 0.6, 2 and 6 μg/mL for the S-DHE) fordosing were achieved by serial dilution of the appropriate stocksolutions which were provided at an approximate concentration of 20μg/mL. Stock solutions were passed through a Millex GV 0.22 μm Duraporesterile filter unit into glass vials and each subsequent dilution withthe sterile citrate buffer was performed by sterile manipulation.Formulations were prepared within the known stability period for (R) DHEand stored refrigerated, at approximately 4° C., until required fordosing.

The reference substance, morphine hydrochloride, was formulated fordosing by dissolving a known amount in citrate buffer to give a 1 mg/mLsolution. A correction factor of 1.32 was applied to enable the dose ofmorphine to be expressed in terms of free-base. Solutions were freshlyprepared and protected from light.

The comparator substance, fentanyl citrate, was formulated for dosing bydissolving a known amount in citrate buffer to give a stock solutionconcentration of 0.36 mg/mL. This stock solution was then seriallydiluted with citrate buffer to give the final concentrations of 0.1, 0.4and 1.2 μg/mL. A correction factor of 1.57 was applied to enable thedose of fentanyl to be expressed in terms of free-base. Solutions werefreshly prepared and protected from light

Group Sizes, Doses and Identification Numbers

There were 11 treatment groups, with up to 10 rats per group. Eachtreatment group was given a letter (A to K). The rats were randomlyallocated to treatment groups on the day prior to dosing based on thepre-dose baseline values for the tail flick test (see below).

D Vehicle 5 mL/kg F R-DHE 0.1 μg/kg E R-DHE 0.3 μg/kg K R-DHE 0.5 μg/kgI S-DHE 3 μg/kg H S-DHE 10 μg/kg G S-DHE 30 μg/kg J Fentanyl 0.5 μg/kg CFentanyl 2 μg/kg B Fentanyl 6 μg/kg A Morphine 5 mg/kg

The vehicle was citrate buffer. The animals were dosed intravenouslyinto a tail vein using a dose volume of 5 mL/kg and a polypropylenesyringe with a Becton Dickinson 25 G (0.5×16 mm) needle. The totalvolume of 5 mL/kg was delivered at as constant a rate as possible over a2 min±10 s interval. The start and stop time for the slow bolus wererecorded. The time of dosing was recorded in the raw data.

Treatment Blinding

Dosing solutions were encoded (A to K) so that the observer did not knowthe identity of the treatment groups.

Body Weights

Animals were weighed prior to testing and body weights recorded on thesame day as the administration of substances.

Procedure

1. Acclimatisation

On one occasion prior to behavioural testing, each animal was subjectedto routine handling and acclimatisation to the behavioural testingenvironment.

2. Baseline Behavioural Testing

The rats were moved to the procedure room 1 day prior to the experiment.The rats were then housed, dosed and observed in the procedure room. Thetail flick test (see below) was performed on all rats on 3 separateoccasions prior to dosing, to establish baseline values. Pre-dosebaseline values were taken as the final test reading (the data from thefirst and second tests were not included but classed as part of theacclimatisation).

Tail flick test: Each rat was lightly held on the surface of the tailflick apparatus (Ugo Basile, Italy), so that its tail was positioneddirectly over the infrared source. The infrared source was then appliedto a small area on the ventral surface of the tail. Activation of theinfrared source simultaneously activated a timer, which automaticallyregistered the time taken to deflect (withdraw or flick) the tail. Thetail flick latency was noted for each animal. The infrared intensity wasset at IR50 and the maximum length of exposure to the IR source was 10s. Non-responding animals were therefore allocated a withdrawal latencyof 10 s.

3. Group Allocation and Exclusion Criteria

Animals were randomly allocated to the treatment groups (A to K) on theday prior to dosing, based on the pre-dose baseline values for the tailflick test.

4. Dosing and Behavioural Testing

The animals were not fasted for this study. Tail flick tests wereperformed approximately 5, 10, 20, 30, 60 and 120 min post-dose (withrespect to the start of dosing), to investigate treatment effect.

5. Terminations

Any animal not allocated to a treatment group was terminated by cervicaldislocation at the end of the study. The remaining animals were returnedto stock on conclusion of the final testing period.

Statistical Analysis

Statistical comparisons were made between DHE (R- and S-isomers),morphine, fentanyl groups with the vehicle group using parametric ornon-parametric statistical procedures. The choice of parametric (one-wayanalysis of variance (ANOVA), Dunnett's t-test) or non-parametric(Kruskal-Wallis statistic, Dunn's test and Mann-Whitney U-test)statistical procedures was based on whether the groups to be comparedsatisfied the homogeneity of variance criterion (evaluated by the LeveneMean test of F-test). Statistical significance was assumed when P<0.05.

In addition, the data were converted to % MPE (Maximum Possible Effect),defined as 100×(test−control)/(cut-off−control) where ‘control’ was thevehicle group observation, ‘test’ was the post-dose observation and‘cut-off’ was the maximum duration of the stimulus allowed (10 s fortail flick). Dose-response curves for each isomer of DHE (R- andS-isomers) and fentanyl were generated for the first 4 observation timepoints and the ED₅₀ (50% MPE dose) was calculated. Analysis wasperformed on the log₁₀ (dose×10³), using a nonlinear regression (line ofbest fit), sigmoidal dose-response. As post-dose data had returned tobaseline at the 60 and 120 min time points, no calculations wererequired on these data.

Results

The group mean±s.e. mean data for tail flick withdrawal latency aresummarised in Table 15. The ED₅₀ values calculated for R-DHE, S-DHE andfentanyl were compared to estimate their relative potencies (Table 11).Time-course graph plots are presented in FIG. 11 to FIG. 13 and ED₅₀(50% MPE dose) dose response curves and data are presented in FIG. 14 toFIG. 17.

TABLE 15 Effects of Dihydroetorphine (R- and S-isomers), fentanyl andmorphine on tail flick withdrawal latency in rats Pre- Tail flicklatency (s) at time (min) post-dose Treatment dose 5 10 20 30 60 120Vehicle 4.2 ± 0.3 5.2 ± 0.6 5.0 ± 0.2 5.1 ± 0.2 4.9 ± 0.4 5.6 ± 0.4 5.1± 0.6 (9) 5 mL/kg i.v. DHE (R-isomer) 4.2 ± 0.3  7.9 ± 0.7* 6.1 ± 0.66.3 ± 0.9 4.7 ± 0.4 4.6 ± 0.5 4.8 ± 0.3   0.1 μg/kg i.v. DHE (R-isomer)4.2 ± 0.3  9.2 ± 0.5**  7.7 ± 0.7^($) 7.6 ± 0.8 6.1 ± 0.9 5.2 ± 0.4 4.6± 0.6   0.3 μg/kg i.v. DHE (R-isomer) 4.4 ± 0.3  9.4 ± 0.6**   9.7 ±0.3^($$$)  8.8 ± 0.5^($$)  8.2 ± 0.8^($)  3.6 ± 0.4** 4.8 ± 0.6 (8) 0.5μg/kg i.v. DHE (S-isomer) 4.2 ± 0.3 8.3 ± 0.8 7.0 ± 0.9 7.0 ± 0.7 5.7 ±0.5 5.8 ± 0.6 4.9 ± 0.4 (9) 3 μg/kg i.v. DHE (S-isomer) 4.2 ± 0.3  9.7 ±0.3^($$)  9.3 ± 0.3^($$) 7.3 ± 0.8 5.8 ± 0.5  4.0 ± 0.4* 3.9 ± 0.4   10μg/kg i.v. DHE (S-isomer) 4.2 ± 0.3   10.0 ± 0.0^($$$)  9.2 ± 0.8^($$)  9.1 ± 0.6^($$$)  8.3 ± 0.7** 4.9 ± 0.3 3.5 ± 0.5 (8) 30 μg/kg i.v.Fentanyl 4.2 ± 0.3 5.8 ± 0.6 5.3 ± 0.6 5.2 ± 0.6 4.6 ± 0.4 4.8 ± 0.4 4.6± 0.4 (9) 0.5 μg/kg i.v. Fentanyl 4.2 ± 0.3  9.0 ± 0.7^($$)   9.1 ±0.4^($$$) 7.5 ± 0.9 7.1 ± 0.7 4.9 ± 0.7 4.2 ± 0.8   2 μg/kg i.v.Fentanyl 4.2 ± 0.3   10.0 ± 0.0^($$$)  8.4 ± 0.7^($$)  8.1 ± 0.7^($) 6.5± 1.0 6.0 ± 1.0 6.3 ± 0.7   6 μg/kg i.v. Morphine 4.2 ± 0.3   10.0 ±0.0^(###)   10.0 ± 0.0^(###)   10.0 ± 0.0^(###)   10.0 ± 0.0^(###)  8.7± 0.9^(#) 6.2 ± 0.9 (8) 5 μg/kg i.v. Vehicle was citrate buffer [citricacid monohydrate:sodium citrate:sodium chloride:water for irrigation inthe ration 0.03:0.10:0.86:99.01 (g:g:g:mL)] Data are expressed as mean ±SEM. N = 10 animals per group, unless otherwise stated in parenthesis.*P < 0.05 and **P < 0.01 when compared to vehicle (ANOVA) and Dunnett'st-test). ^($)P < 0.05, ^($$)P < 0.01 and ^($$$)P < 0.001 when comparedto vehicle (Kruskal-Wallis and Dunn's test). ^(#)P < 0.05 and ^(###)P <0.001 when compared to vehicle (Mann-Whitney U-test).

Effects of Dihydroetorphine (R-DHE) on Tail Flick Withdrawal Latency(FIG. 11)

Intravenous administration of R-DHE at a dose of 0.1 μg/kg caused asignificant increase in the tail flick latency at 5 min post-dose(7.9±0.7 s; P<0.05; ANOVA and Dunnett's t-test) when compared to vehiclegroup data (5.2±0.6 s). Intravenous administration of R-DHE at 0.3 μg/kgcaused a significant increase in the tail flick withdrawal latency at 5and 10 min post-dose (9.2±0.5 s; P<0.01; ANOVA and Dunnett's t-test;7.7±0.7 s; P<0.05; Kruskal-Wallis and Dunn's test, respectively) whencompared to vehicle group data (5.2±0.6 and 5.0±0.2 s, respectively) buthad no effect at any other time points. Intravenous administration ofR-DHE at a dose of 0.5 μg/kg caused a significant increase in the tailflick withdrawal latency at 5, 10, 20 and 30 min post-dose (9.4±0.6 s;P<0.01; ANOVA and Dunnett's t-test; 9.7±0.3 s; P<0.001; 8.8±0.5 sP<0.01; 8.2±0.8 s; P<0.05; all Kruskal-Wallis and Dunn's test,respectively) when compared to vehicle group data (5.2±0.6, 5.0±0.2,5.1±0.2 and 4.9±0.4 s, respectively). A significant decrease observed inthe tail flick latency at 60 min post-dose in the 0.5 μg/kg group is notconsidered to be pharmacologically relevant. No effect was noted at 120min post-dose. These data indicate an immediate analgesic onset, withpeak effects at approximately 5 and 10 min post-dose, returning tobaseline values (comparable to the vehicle control) by the 60 minpost-dose time point.

The estimated ED₅₀ of R-DHE, i.e. the 50% MPE, was 0.08, 0.23, 0.25 and0.42 μg/kg at 5, 10, 20 and 30 min post-dose, respectively. There was nodose response at the 60 and 120 min post-dose time points.

Effects of Dihydroetorphine (S-DHE) on Tail Flick Withdrawal Latency(FIG. 12)

Intravenous administration of S-DHE at a dose of 3 μg/kg did notsignificantly affect tail flick withdrawal latency at any time pointtested when compared to vehicle group data. Intravenous administrationof S-DHE at 10 μg/kg caused a significant increase in the tail flickwithdrawal latency at 5 and 10 min post-dose (9.7±0.3 and 9.3±0.3 srespectively; both P<0.01; Kruskal-Wallis and Dunn's test) when comparedto vehicle group data (5.2±0.6 and 5.0±0.2 s, respectively). Asignificant decrease observed in the tail flick withdrawal latency at 60min post-dose was not considered to be pharmacologically relevant.Intravenous administration of S-DHE at a dose of 30 μg/kg caused asignificant increase in the tail flick withdrawal latency at 5, 10, 20and 30 min post-dose (10.0±0.0 s; P<0.001; 9.2±0.8 s; P<0.01; 9.1±0.6 s;P<0.001; Kruskal-Wallis and Dunn's test and 8.3±0.7 s; P<0.01; ANOVA andDunnett's t-test; respectively) when compared to vehicle group data(5.2±0.6, 5.0±0.2, 5.1±0.2 and 4.9±0.4 s, respectively). These dataindicate an immediate analgesic onset with peak effects at the 5 minpost-dose time point, returning to baseline values (comparable to thevehicle control) by the 60 min post-dose time point.

The estimated ED₅₀ of DHE (S-isomer), i.e. the 50% MPE, was 2.17, 3.80,7.52 and 20.95 μg/kg at 5, 10, 20 and 30 min post-dose, respectively.There was no dose response at the 60 and 120 min post-dose time points.

Effects of Fentanyl on Tail Flick Withdrawal Latency (FIG. 13)

Intravenous administration of fentanyl at a dose of 0.5 μg/kg did notsignificantly affect tail flick withdrawal latency at any time pointtested when compared to vehicle group data. Intravenous administrationof fentanyl at 2 μg/kg resulted in a significant increase in the tailflick withdrawal latency at 5 and 10 min post-dose (9.0±0.7 s; P<0.01and 9.1±0.4 s; P<0.001, respectively; both Kruskal-Wallis and Dunn'stest) when compared to vehicle group data (5.2±0.6 and 5.0±0.2 s,respectively). Intravenous administration of fentanyl at a dose of 6μg/kg caused a significant increase in the tail flick withdrawal latencyat 5, 10 and 20 min post-dose (10.0±0.0 s; P<0.001; 8.4±0.7 s; P<0.01;8.1±0.7 s; P<0.05, respectively; all Kruskal-Wallis and Dunn's test)when compared to vehicle group data (5.2±0.6, 5.0±0.2 and 5.1±0.2 s,respectively). These data indicate an immediate analgesic onset withpeak effects at the 5 min time point, returning to baseline values(comparable to the vehicle control) by the 60 min post-dose time point.

The estimated ED₅₀ of fentanyl, i.e. the 50% MPE, was 1.14, 1.25, 3.11and 9.68 μg/kg at 5, 10, 20 and 30 min post-dose, respectively. Therewas no dose response at 60 and 120 min post-dose time points.

Comparative Effects of R-DHE, S-DHE and Fentanyl

The ED₅₀ values calculated for R-DHE, S-DHE and fentanyl were comparedto estimate their relative potencies (Table 16). The data suggest thatduring the first 30 min, after a single intravenous administration ofeach compound in the male rat, R-DHE had an analgesic potency that is 5-to 23-fold that for fentanyl, S-DHE had an analgesic potency of 0.3- to0.5-fold that of fentanyl, and that R-DHE has an analgesic potency thatis 17- to 50-fold that for S-DHE.

TABLE 16 ED₅₀ values and comparative ratios of R-DHE, S-DHE and fentanylTime ED₅₀ ratio Post- ED₅₀ ED₅₀ ED₅₀ ED₅₀ ratio ED₅₀ ratio fentanyl/dose R-DHE S-DHE fentanyl fentanyl/ fentanyl/ S-DHE/ (min) (μg/kg)(μg/kg) (μg/kg) R-DHE S-DHE R-DHE 5 0.08 2.17 1.14 14 0.5 28 10 0.233.80 1.24 5 0.3 17 20 0.25 7.52 3.11 12 0.4 30 30 0.42 20.95 9.68 23 0.550

Effects of Morphine on Tail Flick Withdrawal Latency

Intravenous administration of morphine (5 mg/kg) caused a significantincrease in the tail flick withdrawal latency at 5, 10, 20, 30 (10.0±0.0s; P<0.001; Mann-Whitney U-test, for all 4 time points) and 60 minpost-dose (8.7±0.9 s; P<0.05; Mann-Whitney U-test) when compared tovehicle group data (5.2±0.6, 5.0±0.2, 5.1±0.2, 4.9±0.4 and 5.6±0.4 s,respectively).

Conclusion

A single intravenous administration of R-DHE at doses of 0.1, 0.3 and0.5 μg/kg and S-DHE at doses of 10 and 30 μg/kg caused a significantdose-dependent increase in the tail flick withdrawal latency of malerats up to 30 min post-dose. Intravenous administration of fentanyl atdoses of 2 and 6 μg/kg caused a significant dose-dependent increase intail flick withdrawal latency up to 30 min post-dose.

The ED₅₀ values calculated for R-DHE, S-DHE and fentanyl were comparedto estimate their relative potencies (Table 16). The data suggest thatduring the first 30 min after a single intravenous administration ofeach compound in the male rat that; R-DHE had an analgesic potency thatis 5- to 23-fold that for fentanyl, S-DHE had an analgesic potency of0.3- to 0.5-fold that of fentanyl, and that R-DHE has an analgesicpotency that is 17- to 50-fold that for S-DHE.

The duration of opioid analgesic activity of R-DHE and S-DHE followingintravenous administration highlights the potential benefit andtherapeutic potential of these compounds in the treatment of acute pain.

The effects noted following administration of morphine are consistentwith its known pharmacological activity and thus this test system wassensitive to detect nociceptive effects.

Effects of (R) and (S)-Dihydroetorphine in the Spinal Nerve LigationModel of Neuropathic Pain

The test model used is well known in the art and is described in Pain1992; 50: 355-363 (Kim S H, Chung J M, An experimental model forperipheral neuropathy produced by segmental spinal nerve ligation in therat).

The potential analgesic effects of dihydroetorphine following a singleintravenous dose of 0.1, 0.3 and 0.5 μg/kg (R isomer) and a singleintravenous dose of 3, 10 and 30 μg/kg (S-isomer) in the spinal nerveligation model of neuropathic pain was investigated. A peripheralmononeuropathy was induced in the left hind limb of rats by tightligation of the L5 and L6 spinal nerves. The development of mechanicalallodynia and thermal hyperalgesia was monitored using establishedbehavioural tests (Von Frey test and the Hargreaves Plantar testrespectively). Morphine was used as a reference substance and Pregabalinwas used as a comparator substance.

Test Substances and Materials

Test Substances, Reference Substance, Comparator Substance and Vehicles

Test substances: Dihydroetorphine (R-isomer) and Dihydroetorphine(S-isomer) Vehicle for test and reference substances:

Citrate buffer (citric acid monohydrate:sodium citrate:sodiumchloride:sterile water, in the ratio 0.03:0.10:0.86:99.01 (g:g:g:mL);[citric acid monohydrate (white powder; Sigma, UK), sodium citrate(Sigma, UK), sodium chloride (white solid; Merck), sterile water (clearliquid; Baxter Healthcare, UK)]

Reference substance: Morphine hydrochloride (white powder; MacfarlanSmith, Edinburgh, UK)

Comparator substance: Pregabalin (Trade name Lyrica®; white capsules;manufactured by Pfizer and supplied by Lindsay & Gilmour Chemist,Juniper Green, Edinburgh)

Vehicle for comparator substance: 1% w/v Carboxy methylcellulose (CMC,powder; Sigma, UK)

Test, Reference and Comparator Substance Storage

The test substances were stored at room temperature, protected fromlight and the reference and comparator substances were stored at roomtemperature,

Route of Administration and Dose Levels

The route of administration of R- and S-isomer forms of dihydroetorphineand the vehicle (citrate buffer) was intravenous. This is a potentialroute of administration in humans. The doses of R-DHE were 0.1, 0.3 and0.5 μg/kg and the doses of S-DHE were 3, 10 and 30 μg/kg.

The route of administration of morphine was intravenous. The dose ofmorphine was 5 mg/kg.

The route of administration of the comparator substance Pregabalin wasoral. In Phase 2 of the study, the dose of Pregabalin, was 30 mg/kg. ForPhase 3, the dose levels of the comparator substance, Pregabalin, were30, 50 and 100 mg/kg.

Animals

Species: Rat

Strain: Sprague-Dawley

Sex: Male

Number of animals. 75 animals were surgically prepared.

Age range: 6 to 7 weeks (for surgery); 8 to 9 weeks (dosing Phase 1); 9to 10 weeks (dosing Phase 2); 11 to 12 weeks (dosing Phase 3).

Weight range: 139 to 183 g (for surgery); 190 to 257 g (dosing Phase 1);210 to 284 g (dosing Phase 2); 243 to 341 g (dosing Phase 3).

Acclimatisation: 3 days after delivery, before commencing thebehavioural testing

Source: Harlan UK Ltd

Animal Identification and Randomisation

Each animal was arbitrarily allocated a unique identification numberwhich appeared on the data sheets and cage cards. Animals wereidentified by a waterproof tail mark.

Animal Health and Welfare

All studies were conducted in accordance with the legislation under theAnimals (Scientific Procedures) Act 1986, with UK Home Office Guidanceon the implementation of the Act and with all applicable Codes ofPractice for the care and housing of laboratory animals.

Housing and Environment

Animals were housed in groups of up to 5 in sawdust filled solid-bottomcages. During the acclimatisation, the rooms and cages were cleaned atregular intervals to maintain hygiene. The rooms were illuminated byfluorescent lights set to give a 12 h light-dark cycle (on 07.00, off19.00), as recommended in the Home Office Animals (ScientificProcedures) Act 1986. The rooms were air-conditioned and the airtemperature and relative humidity measured. During the acclimatisationperiod room temperature was maintained (range 20° C. to 22° C.) andhumidity levels were within the range 46% to 59%. The procedure roomtemperature was maintained (range 19° C. to 22° C.) and humidity levelswere within the range 26% to 43%.

Diet and Water

An expanded rodent diet of RM1(E) SQC (Special Diets Services, Witham,UK) and mains tap water were offered ad libitum. Each batch of diet wasdelivered with an accompanying certificate of analysis (C of A)detailing nutritional composition and levels of specified contaminants(e.g. heavy metals, aflatoxin and insecticides). The water wasperiodically analysed for impurities and contaminants. The criteria foracceptable levels of contaminants in stock diet and water supply werewithin the analytical specifications established by the dietmanufacturer and water analytical service, respectively.

Health Status

The animals were examined on arrival and prior to the study; all animalswere healthy and considered suitable for experimental use.

Experimental Design

Formulation of the Test, Reference and Comparator Substances

The citrate buffer was prepared by accurately weighing the appropriatequantities of each component and dissolving them in sterile water forinjection. When the components were fully dissolved the osmolality andpH of the solution was measured. The vehicle was deemed acceptable asthe pH was 5.03, which was within the range 4.8 to 5.2, and theosmolality was 295 mOsmol/kg between the range of 280 to 300 mOsmol/kg.The vehicle was then terminally filtered through a Millex GV stericup(0.22 μm filter) under aseptic conditions and stored at 2° C. to 8° C.prior to use.

The test substances, dihydroetorphine (R- and S-isomers), wereformulated for dosing as solutions in citrate buffer. The desiredconcentrations (0.02, 0.06 and 0.10 μg/mL for the R isomer and 0.6, 2and 6 μg/mL for the S-isomer) for dosing were achieved by serialdilution of the appropriate stock solutions which were provided at anapproximate concentration of 20 μg/mL. The actual concentration of thestock solutions was noted in the raw data. Prior to serial dilution,stock solutions were passed through a Millex GV 0.22 μm Durapore sterilefilter unit into glass vials and each subsequent dilution with thesterile citrate buffer was performed by sterile manipulation. Nocorrection factor was applied and formulations were prepared as the freebase. Formulations were prepared in advance of the study dosing datesand were used (1-2 days following preparation) within the knownstability for R-DHE which was 11 days. S-DHE was used 1-2 days followingpreparation. Dosing solutions of dihydroetorphine (R- and S-isomers)were stored refrigerated, at approximately 4° C., until they wererequired for dosing.

The reference substance, morphine hydrochloride, was formulated fordosing by dissolving a known amount in citrate buffer to give a 1 mg/mLsolution. A correction factor of 1.32 was applied to enable the dose ofmorphine to be expressed in terms of free base. Solutions were freshlyprepared and protected from light.

The comparator substance, Pregabalin, was formulated for dosing bysuspending a known amount in 1% w/v CMC to give a 3 mg/mL suspension forPhase 2 and 3, 5 and 10 mg/mL suspensions were prepared for Phase 3. Nocorrection factor was required therefore the Pregabalin was dosed as afree-base. Suspensions were freshly prepared and protected from light.

Group Sizes, Doses and Identification Numbers

There were 5 treatment groups, with a maximum of 10 rats per group. Eachtreatment group was given a letter (Phase 1: A to E, Phase 2: F to J andPhase 3: K to O). The rats were randomly allocated to treatment groupson the day prior to dosing based on the pre dose baseline values for thethermal hyperalgesia test (see below).

Phase 1: C Vehicle 5 mL/kg, i.v. B R-DHE 0.1 μg/kg, i.v. A R-DHE 0.3μg/kg, i.v. E R-DHE 0.5 μg/kg, i.v. D Morphine 5 mg/kg, i.v. Phase 2: IVehicle 5 mL/kg, i.v. F S-DHE 3 μg/kg, i.v. G S-DHE 10 μg/kg, i.v. JS-DHE 30 μg/kg, i.v. H Pregabalin 30 mg/kg, p.o. Phase 3: N Vehicle 10mL/kg, p.o. M Pregabalin 30 mg/kg, p.o. L Pregabalin 50 mg/kg, p.o. KPregabalin 100 mg/kg, p.o. O Morphine 5 mg/kg, i.v.

The intravenous vehicle, citrate buffer, was used for Phases 1 and 2 andthe oral vehicle, 1% w/v CMC, was used for Phase 3. Animals allocated tointravenous treatment groups were dosed into a tail vein using a dosevolume of 5 mL/kg and a polypropylene syringe with a Becton Dickinson 25G (0.5×16 mm) needle. The total intravenous volume of 5 mL/kg wasdelivered at as constant a rate as possible over a 2 min±10 s interval.The start and stop time for the slow intravenous bolus was recorded.Animals allocated to oral treatment groups were dosed by oral gavage,using a dose volume of 10 mL/kg. The time of dosing was recorded in theraw data.

Treatment Blinding

Dosing solutions were encoded (Phase 1: A to E, Phase 2: F to J andPhase 3: K to O) so that the observers were not aware of the identity ofthe treatment groups. As the comparator substance in Phase 2 of thestudy was administered by a different dose route, this group was notblinded to the person performing the dosing and was encoded H. Also, asthe morphine control in Phase 3 was administered intravenously, and thiswas a different route to the vehicle and comparator substance groups(oral dosing), the morphine group was not blinded, and was thereforeencoded 0.

Body Weights

Animals were weighed prior to surgery, on Day 1 post-operatively (PO),and on each day of dosing prior to administration of substances, andbody weights were recorded.

Daily Observations

General observations were made on all animals on a daily basis from Day0 PO onwards, with particular attention being paid to the condition ofthe animal's left and right hind paws.

Procedure

1 Acclimatisation

Prior to behavioural testing, the animals were subjected to routinehandling and acclimatisation to the behavioural testing environment.

2 Baseline Behavioural Testing

The rats were moved to the procedure room 1 day prior to the experiment.The rats were then housed, dosed and observed in the procedure room. Thebehavioural tests (see below) were performed on all rats on 2 separateoccasions prior to surgery, to establish baseline values. Pre-surgerybaseline values were taken as the data from the final (second) day oftesting (the data from the first day of testing was classified as partof the acclimatisation). The sequence of tests was mechanical allodyniafollowed by thermal hyperalgesia, with a minimum 5 min period allowedbetween the tests.

Mechanical allodynia (Von Frey test): Each animal was placed in a wiremesh cage and a series of Von Frey filaments applied to the plantarsurface of the hind paw, from below. The filaments were applied inascending order (starting with the weakest force), and the withdrawalthreshold for both the left and right hind paws was evaluated. Eachfilament was indented on the mid-plantar surface of the foot to thepoint where it just started to bend; this was repeated approximately 8to 10 times per filament at a frequency of approximately 1 Hz. Thewithdrawal threshold was defined as being the lowest force of two ormore consecutive Von Frey filaments to elicit a reflex withdrawalresponse (i.e. a brief paw flick).

Thermal hyperalgesia (Hargreaves Plantar test): Each rat was placed in aclear plastic chamber with a glass floor and allowed a short period toacclimatise to their environment prior to testing (approximately 1 min).The animals were then challenged with a radiant infrared (IR) heatsource, directed at the plantar surface of their hind paw from below,and the withdrawal latency calculated for both the left and right hindpaws. The infrared intensity was set at IR50 (setting designed todeliver a heat flux reading of 250 mW cm³) and the maximum length ofexposure to the IR source was 18 s. Non responding animals weretherefore allocated a withdrawal latency of 18 s.

3 Surgical Procedure

The animals were surgically prepared over 3 days. Each rat wasanaesthetised as necessary with isofluorane in 1% to 3% oxygen. Each ratwas placed in a prone position and the surface around the incision sitewas shaved and sterilised with surgical spirit. Under asepticconditions, the left paraspinal muscles were separated from the spinousprocesses at L4 S2 levels. The L6 transverse process was then carefullyremoved with a small rongeur and the L4 L6 spinal nerves identified. Theleft L5 and L6 spinal nerves were isolated, and tightly ligated using6-0 silk thread (as viewed under ×40 magnification). The overlyingmuscle and skin were closed in layers using appropriate suture material,and once complete, the anaesthesia was discontinued. On recovery fromanaesthesia, rats were re-housed with their cage-mates, initially onsoft padded bedding overnight to reduce the risk of infection, andsubsequently on sawdust bedding following full recovery. The animalswere allowed to recover for a minimum of 4 days before the behaviouraltesting was recommenced.

4 Developmental Testing

Following surgery, behavioural testing was conducted twice prior to thedosing day, to monitor the development of allodynia/hyperalgesia.Additional discretionary testing days were also included to ensure thata sufficient number of animals had developed allodynia hyperalgesiaprior to each dosing phase.

5 Group Allocation and Exclusion Criteria

Animals were randomly allocated to the treatment groups on the day priorto dosing based on the pre dose baseline values for the thermalhyperalgesia test. Only animals which developed both mechanicalallodynia and thermal hyperalgesia were included in the study. Animalswere deemed to have developed mechanical allodynia if their left pawwithdrawal threshold to Von Frey filaments was 5 g of force (whichcorresponds to monofilament number 4.56 or less). Animals were deemed tohave developed thermal hyperalgesia if their left paw withdrawal latencyto the thermal plantar device showed ≧30% difference from the left pawpre-surgery value, prior to dosing.

6 Dosing and Behavioural Testing

The animals were not fasted for this study. On each day of dosing, theallocated animals each received a single intravenous dose of testsubstance, reference substance or vehicle; or an oral dose of comparatorsubstance or vehicle. There were 3 phases to the study. The dosing ofeach phase was split over 2 days and animals from a minimum of 3treatment groups were dosed on each dosing day. Following completion ofeach phase, the animals were allowed a minimum washout period of 1 weekbefore commencing the subsequent phase of the study.

In Phases 1 and 2 of the study, following dosing (approximately 5 minfrom the start of dosing) and at approximately 25, 50 and 120 minpost-dose, the left and right hind paw of each rat was assessed formechanical allodynia using the Von Frey test. At approximately 15, 35,60 and 130 min post-dose, the left and right hind paw of each rat wasassessed for thermal hyperalgesia using the Hargreaves Plantar test, toinvestigate treatment effect. All time points were with respect to thestart of dosing.

In Phase 3 of the study, at approximately 60, 120, 180 and 240 minpost-dose the left and right hind limb of each rat were assessed formechanical allodynia using the Von Frey test. At approximately 70, 130,190 and 250 min post-dose, the left and right hind limb of each rat wereassessed for thermal hyperalgesia using the Hargreaves Plantar test, toinvestigate treatment effect.

7 Terminations

All study animals were terminated by cervical dislocation followingconclusion of the final testing period.

Statistical Analysis

Statistical comparisons were made between treatment groups usingparametric (e.g. one way analysis of variance, Dunnett's t-test,Student's t-test) or non-parametric (e.g. Kruskal-Wallis statistic,Dunn's test, Mann-Whitney U-test) statistical procedures. The choice ofparametric or non-parametric test was based on whether the groups to becompared satisfy the homogeneity of variance criterion (evaluated by theLevene Mean test or F-test). The Von Frey data was logarithmicallytransformed (log 10 of (force in grams×10 000)) prior to analysis. InPhase 2 of the study, as the comparator substance, Pregabalin wasadministered by a different dose route, the data for Pregabalin werecompared to the pre-dose values using a paired Student's t test. InPhase 3 of the study, as the reference substance, Morphine wasadministered by a different dose route to the vehicle and testsubstance, the Morphine data were compared to the pre-dose values usinga paired Student's t-test. For all tests, statistical significance wasassumed when P<0.05. The statistical significance for the von Frey test,although performed on the logarithmically transformed data, wasexpressed with respect to the grams force in the results section forillustration purposes. Full details of the analysis are given in the rawdata.

Results

The group mean±s.e. mean data for withdrawal thresholds and withdrawallatency are summarised in Table 17 to Table 22.

Development of Neuropathic Pain States

Two different components of neuropathic pain were investigated usingestablished behavioural tests, namely Von Frey filaments to test for thepresence of mechanical allodynia, and the Hargreaves Plantar test totest for the presence of thermal hyperalgesia. The majority of animalswhich underwent a spinal nerve ligation, exhibited a marked increase insensitivity of the left hind paw to the two behavioural tests in thedays post-injury, indicative of the development of a peripheralmononeuropathy. The right hind paw showed no increase in sensitivitypost surgery. In each Phase of the study, all animals dosed were deemedto have neuropathy in the left hind paw as assessed using theestablished behavioural tests the day prior to dosing.

Effects of R-DHE on Behavioural Test Responses (Phase 1)

Mechanical allodynia: In Phase 1, intravenous administration of R-DHE at0.1 and 0.3 μg/kg did not produce any significant changes in left orright paw withdrawal thresholds to Von Frey filaments. However,intravenous administration of R-DHE at 0.5 μg/kg, caused a significantincrease in the left paw withdrawal threshold at approximately 5 minpost-dose (21.97±2.30 g; P 0.01; Kruskal-Wallis and Dunn's test) whencompared to vehicle group values of 5.43±2.58 g, and also atapproximately 25 min post-dose (13.12±3.41 g; P<0.05; ANOVA andDunnett's t-test) when compared to vehicle group values of 2.25±0.75 g(Table 17, FIG. 18).

Thermal hyperalgesia: Intravenous administration of R-DHE failed to havea significant effect on left paw withdrawal latencies when compared tovehicle. Intravenous administration of R-DHE at a dose of 0.3 μg/kgcaused a significant increase in the right paw withdrawal latency atapproximately 35 min post-dose (14.5±0.7 s; P<0.05; ANOVA and Dunnett'st-test) when compared to vehicle values (10.3±0.9 s); however, this isphysiologically irrelevant as the withdrawal threshold in the right pawwas similar to the pre dose values for the right paw (14.3±0.6 s) (Table18, FIG. 19).

Effects of S-DHE on Behavioural Test Responses (Phase 2)

Mechanical allodynia: In Phase 2, intravenous administration of S-DHE at3 and 10 μg/kg did not produce any significant changes in left or rightpaw withdrawal thresholds to Von Frey filaments. However, intravenousadministration at 30 μg/kg caused a significant increase in the left pawwithdrawal threshold at approximately 5 min post-dose (24.56±0.33 g;P≦0.001; Kruskal-Wallis and Dunn's test) when compared to vehicle groupvalues of 6.11±2.39 g, and also at approximately 25 min post-dose(21.92±1.70 g; P<0.001; Kruskal Wallis and Dunn's test) when compared tovehicle group values of 1.66±0.47 g (Table 19, FIG. 20).

Thermal hyperalgesia: Intravenous administration of S-DHE failed to havea significant effect on left and right paw withdrawal latencies at dosesof 3 and 10 μg/kg when compared to the vehicle group values. Intravenousadministration of S-DHE at a dose of 30 μg/kg, however, did produce asignificant increase in the paw withdrawal latencies at approximately 15min post-dose in both left (17.6±0.4 s; P<0.01; Kruskal-Wallis andDunn's test) and right (17.5±0.4 s; P<0.01; Kruskal-Wallis and Dunn'stest) hind paws, when compared to vehicle values of 10.3±0.8 s and13.4±0.8 s, respectively. The increase in right paw latency may beindicative of the central effects of dihydroetophine (S-isomer) at the30 μg/kg dose level (Table 20, FIG. 21).

Effects of Morphine on Behavioural Test Responses (Phases 1 and 3)

In Phase 1, the morphine reference was compared to vehicle as both wereadministered orally. In Phase 3, the morphine reference was compared topre-dose as it was not be relevant to compare this to an oral vehicle).

Mechanical allodynia: Following intravenous administration of morphineat 5 mg/kg (Phase 1), the left hind paw withdrawal thresholdsignificantly increased at approximately 25 min post-dose (19.23±2.73 g;P<0.001; unpaired 2-tailed Student's Mast) and approximately 50 minpost-dose (21.55±2.40 g; P<0.001; unpaired 2-tailed Student's t test)when compared to vehicle values of 2.25±0.75 and 2.11±0.82 g. There wasa significant decrease observed in the right paw pre dose data(16.37±2.20 g; P<0.05; unpaired 2-tailed Student's t-test) when comparedto vehicle values (22.26±1.52 g). This was unavoidable, as theallocation to treatment groups was based on the pre-dose values from thethermal hyperalgesia test (Table 17, FIG. 18). No other significanteffects were noted in the right hind paw.

In Phase 3, intravenous administration of morphine (5 mg/kg)significantly increased the left paw withdrawal thresholds atapproximately 60 min (21.32±2.56 g; P<0.001; paired 2 tailed Student'st-test), 120 min (11.08±2.85 g; P<0.01; paired 2-tailed Student'st-test) and approximately 180 min post-dose (3.68±0.97 g; P<0.05; paired2-tailed Student's t test) compared to pre-dose values 1.46±0.37 g(Table 21, FIG. 22).

Thermal hyperalgesia: In Phase 1, intravenous administration of morphineat 5 mg/kg caused a significant increase in the withdrawal latencies inthe left paw across all of the time points tested and in the right pawat approximately 15, 35 and 60 min post-dose. At approximately 15 minpost-dose; (left; 12.0±1.5 s; P<0.05; Mann-Whitney U-test), (right;17.5±0.5 s; P<0.001; Mann-Whitney U-test), at 35 min post-dose (left;16.4±0.9 s; P<0.001; unpaired 2-tailed Student's t-test), (right;16.8±0.7 s; P<0.001; unpaired 2-tailed Student's t-test) and atapproximately 60 min post-dose in both paws (left; 12.8±1.3 s; P<0.01;unpaired 2 tailed Student's t-test), (right; 16.3±1.1 s; P<0.05;unpaired 2-tailed Student's t-test) and approximately 130 min post-dosein the left paw (10.6±0.9 s; P<0.05; unpaired 2-tailed Student's t-test)when compared to vehicle values (7.5±0.5, 12.8±1.1, 7.0±1.2, 10.3±0.9,7.6±0.9, 12.4±1.4 and 7.4±0.9 s, respectively) (Table 18, FIG. 19).

In Phase 3 of the study, intravenous administration of morphine caused asignificant increase in left paw withdrawal latency at approximately 70min post-dose (11.8±1.2 s; P<0.01; paired 2-tailed Student's t-test),approximately 190 min post-dose (8.0±0.8 s; P<0.05; paired 2-tailedStudent's t-test) and approximately 250 min post-dose (10.6±1.4 s;P<0.05; paired 2-tailed Student's t-test) when compared to pre-dosevalues of 6.0±0.5 s (Table 22, FIG. 23).

Effects of Pregabalin on Behavioural Test Responses (Phases 2 and 3)

Pregabalin was compared to pre-dose in Phase 2 but compared to vehiclein Phase 3 of the study. In Phase 2 of the study the Pregabalin wasadministered by a different route (oral) to the vehicle (iv), so acomparison with vehicle was not appropriate. In Phase 3 of the study thedose response to Pregabalin, using 3 dose levels was compared to vehiclerather than pre dose.

Mechanical allodynia: In Phase 2, oral administration of Pregabalin (30mg/kg) caused a significant increase in the left paw withdrawalthreshold at approximately 50 min post-dose (10.35±2.51 g; P<0.001;paired 2-tailed Student's t-test) and approximately 120 min post dose(13.90±3.00 g; P<0.001; paired 2-tailed Student's t-test) when comparedto pre dose values of 1.09±0.35 g. (Table 19, FIG. 20).

In Phase 3, Pregabalin, administered orally, at a dose of 30 mg/kgcaused a significant increase in the left paw withdrawal threshold atapproximately 120 min post-dose (17.06±2.88 g; P<0.01; Kruskal-Wallisand Dunn's test) and approximately 180 min post dose (13.86±3.21 g;P<0.01; ANOVA and Dunnett's test) when compared to vehicle values of5.00±2.34 g and 2.57±0.92 g, respectively. Oral administration ofPregabalin at a dose of 50 mg/kg caused a significant increase and bothleft and right paw withdrawal thresholds at approximately 180 minpost-dose (left paw: 15.20±3.31 g; P<0.01; ANOVA and Dunnett's test andright paw: 24.20±0.39 g; P<0.05; Kruskal-Wallis and Dunn's test) whencompared to vehicle values of 2.57±0.92 g and 16.57±1.75 g,respectively, and on the left paw withdrawal threshold at approximately240 min post-dose (12.05±3.41 g; P<0.05; Kruskal-Wallis and Dunn's test)when compared to vehicle values (1.48±0.30 g). Oral administration ofPregabalin at a dose of 100 mg/kg caused a significant increase in leftpaw withdrawal threshold at approximately 120 min post-dose (23.29±1.19g; P<0.01; ANOVA and Dunnett's test), in left and right paws atapproximately 180 min post-dose (left paw: 19.77±2.70 g; P<0.01; ANOVAand Dunnett's test and right paw: 23.70±1.04 g; P<0.01; Kruskal-Wallisand Dunn's test) when compared to vehicle values of 2.57±0.92 and16.57±1.75 g, respectively, and at approximately 240 min post-dose inthe left paw withdrawal threshold (15.91±2.86 g; P<0.001; Kruskal-Wallisand Dunn's test) when compared to vehicle values (1.48±0.30 g). Theincrease in the right paw withdrawal thresholds following administrationof Pregabalin at 50 and 100 mg/kg, was indicative of the central effectsof Pregabalin at these dose levels. This was consistent with the animalsdisplaying a dose-dependant increase in the level of sedative clinicalsymptoms. (Table 21, FIG. 22).

Thermal hyperalgesia: In Phase 2, oral administration of Pregabalin (30mg/kg) caused a significant increase in the left paw withdrawal latencyat approximately 15, 35, 60 and 130 min post-dose (8.3±0.7 s P<0.05;8.6±1.0 s P<0.05; 8.8±1.0 s P<0.05; 9.6±0.8 s P<0.001; all paired2-tailed Student's t-test) when compared to the pre dose value of6.2±0.5 s. These significant increases were not deemed to bepharmacologically relevant, as there were similar increases in thevalues for withdrawal latencies in the vehicle control group (ivadministration) in Phase 2 (approximately 15, 35, 60 and 130 minpost-dose (10.3±0.8 s P<0.01; 8.1±0.5 s P<0.05; 9.3±0.7 s P<0.001;9.8±1.0 s P<0.01; all paired 2-tailed Student's t-test) when compared tothe pre dose value of 6.2±0.5 s), and in Phase 3 of the study, oraladministration of Pregabalin failed to have a significant effect in leftand right paw withdrawal latencies, at all of the doses tested (30, 50and 100 mg/kg) and across all post-dose time points (60, 120, 180 and240 min), when compared to the vehicle control. (Table 20, FIG. 21 andTable 22, FIG. 23).

Conclusion

A peripheral mononeuropathy was induced in the left hind limb of rats bytight ligation of the L5 and L6 spinal nerves. The development ofmechanical allodynia and thermal hyperalgesia was monitored usingestablished behavioural tests (Von Frey test and the Hargreaves Plantartest, respectively). Response threshold and latency was assessed forboth the left (affected) and right (unaffected) hind paws. In each Phaseof the study, all animals dosed were deemed to have neuropathy in theleft hind paw as assessed using the established behavioural tests theday prior to dosing.

Intravenous administration of R-DHE at 0.5 μg/kg caused an increase inwithdrawal threshold (mechanical allodynia) of up to 25 min post-dosewith peak effects at approximately 5 min post-dose. There were noeffects of R-DHE at 0.5 μg/kg on withdrawal latency (thermalhyperalgesia) at any of the time points tested. There were also noeffects on either mechanical allodynia or thermal hyperalgesia at thelower doses of 0.1 and 0.3 μg/kg of R-DHE.

Intravenous administration of S-DHE at a dose of 30 μg/kg causedsignificant analgesic effects in both withdrawal threshold (mechanicalallodynia), with peak effects at approximately 5 to 25 min post-dose andwithdrawal latency (thermal hyperalgesia), with peak effect atapproximately 15 min post dose. No effects on mechanical allodynia orthermal hyperalgesia were noted at 3 and 10 μg/kg of S-DHE.

Intravenous administration of the opioid compounds, R-DHE and S-DHEdemonstrated analgesic activity in both the mechanical allodynia andthermal hyperalgesia test in the rat. This highlights the therapeuticpotential of these compounds in the treatment of neuropathic pain.

Following administration of Pregabalin (in Phase 3) at doses up to 100mg/kg, there was a dose dependent increase in withdrawal threshold withpeak effects between approximately 180 and 240 min post-dose. There wereno effects of pregabalin in the thermal hyperalgesia test. The effectsnoted following administration of Pregabalin were consistent with itsknown pharmacological activity (based on literature data) withsignificant effects on mechanical allodynia, but a limited effect onthermal hyperalgesia.

The effects noted following intravenous administration of morphine wereconsistent with its known pharmacological activity with significanteffects on mechanical allodynia and thermal hyperalgesia. This testsystem was therefore sensitive to detect nociceptive effects in both themechanical allodynia and the thermal hyperalgesia test in the rat.

TABLE 17 Effects of intravenous R-DHE on the left (L) and right (R) pawwithdrawal thresholds to Von Frey monofilament challenges in rats(Phase 1) (a) Raw Data Withdrawal Threshold (g) at Time (min) Post-DosePhase 1 Pre-Dose 5 25 Treatment L R L R L R Vehicle † 0.95 ± 0.09 22.26± 1.52  5.43 ± 2.58 22.26 ± 1.52  2.25 ± 0.75 23.25 ± 1.16 5 mL/kg, i.v.(9) (9) (9) (9) (9) (9) Dihydroetorphine 1.37 ± 0.31 21.07 ± 1.99  5.35± 0.79 23.30 ± 1.00  6.55 ± 3.04 19.81 ± 1.97 (R) 0.1 μg/kg, i.v.Dihydroetorphine 1.27 ± 0.33 17.45 ± 2.01 11.13 ± 3.54 22.20 ± 1.30 2.41 ± 0.59 22.37 ± 1.36 (R) 0.3 μg/kg, i.v. Dihydroetorphine 1.05 ±0.18 16.32 ± 1.99 21.97 ± 2.30 24.36 ± 0.34 13.12 ± 3.41  24.36 ± 0.34(R) 0.5 μg/kg, i.v. Morphine 1.69 ± 0.53 16.37 ± 2.20 13.38 ± 3.14 23.50± 1.02 19.23 ± 2.73  23.50 ± 1.02 5 mg/kg, i.v. (a) Raw Data WithdrawalThreshold (g) at Time (min) Post-Dose Phase 1 50 120 Treatment L R L RVehicle †  2.11 ± 0.82 22.26 ± 1.52 1.09 ± 0.23 23.25 ± 1.16 5 mL/kg,i.v. (9) (9) (9) (9) Dihydroetorphine  1.35 ± 0.32 18.11 ± 2.52 1.07 ±0.16 18.05 ± 1.94 (R) 0.1 μg/kg, i.v. Dihydroetorphine  1.14 ± 0.3118.55 ± 2.25 0.95 ± 0.10 18.25 ± 1.54 (R) 0.3 μg/kg, i.v.Dihydroetorphine  6.17 ± 2.33 21.84 ± 1.77 1.80 ± 0.88 20.41 ± 1.63 (R)0.5 μg/kg, i.v. Morphine 21.55 ± 2.40 24.56 ± 0.33 9.37 ± 3.60 23.46 ±1.06 5 mg/kg, i.v. (b) Log Data Withdrawal Threshold (Log 10 (force (g)× 10 000)) at Time (min) Post-Dose Phase 1 Pre-Dose 5 25 Treatment L R LR L R Vehicle † 3.96 ± 0.04 5.34 ± 0.03 4.48 ± 0.16 5.34 ± 0.03 4.21 ±0.12 5.36 ± 0.03 5 mL/kg, i.v. (9) (9) (9) ^($) (9) (9) (9)Dihydroetorphine 4.06 ± 0.08 5.30 ± 0.05 4.65 ± 0.10 5.36 ± 0.02 4.42 ±0.19 5.27 ± 0.05 (R) 0.1 μg/kg, i.v. Dihydroetorphine 4.01 ± 0.09 5.21 ±0.05 4.71 ± 0.20 5.34 ± 0.03 4.26 ± 0.10 5.34 ± 0.03 (R) 0.3 μg/kg, i.v.Dihydroetorphine 3.96 ± 0.07 5.18 ± 0.05 5.26 ± 0.12 ^(##) 5.39 ± 0.014.89 ± 0.17 ^(‡) 5.39 ± 0.01 (R) 0.5 μg/kg, i.v. Morphine 4.08 ± 0.115.17 ± 0.06 * 4.93 ± 0.16 5.37 ± 0.02 5.20 ± 0.11 *** 5.37 ± 0.02 5mg/kg, i.v. (b) Log Data Withdrawal Threshold (Log 10 (force (g) × 10000)) at Time (min) Post-Dose Phase 1 50 120 Treatment L R L R Vehicle †4.12 ± 0.14 5.34 ± 0.03 3.96 ± 0.08 5.36 ± 0.03 5 mL/kg, i.v. (9) (9)(9) (9) Dihydroetorphine 4.05 ± 0.08 5.21 ± 0.07 3.99 ± 0.06 5.23 ± 0.05(R) 0.1 μg/kg, i.v. Dihydroetorphine 3.97 ± 0.08 5.23 ± 0.06 3.95 ± 0.055.25 ± 0.04 (R) 0.3 μg/kg, i.v. Dihydroetorphine 4.50 ± 0.17 5.32 ± 0.054.03 ± 0.12 5.30 ± 0.03 (R) 0.5 μg/kg, i.v. Morphine 5.26 ± 0.11 ***5.39 ± 0.00 4.49 ± 0.23 5.36 ± 0.02 5 mg/kg, i.v. Data are expressed asMean ± SEM. n = 10 animals per group, unless stated in parenthesis.Statistical analysis only performed on log data. † Vehicle = citratebuffer (citric acid monohydrate:sodium citrate:sodium chloride:water forinjection, in the ratio, 0.03:0.10:0.86:99.01 (g:g:g:mL). * P < 0.05when compared to vehicle group data (unpaired 2-tailed Student'st-test). *** P < 0.001 when compared to vehicle group data (unpaired2-tailed Student's t-test). ^(##) P < 0.01 when compared to vehiclegroup data (Kruskal-Wallis and Dunn's test). ^(‡) P < 0.05 when comparedto vehicle group data (ANOVA and Dunnett's t-test). ^($) P < 0.05 whencompared to pre-dose data (paired 2-tailed Student's t-test).

TABLE 18 Effects of intravenous R-DHE on the left (L) and right (R) pawwithdrawal latency to a thermal plantar stimulus in rats (Phase 1)Withdrawal Latency (s) at Time (min) Post-Dose Phase 1 Pre-Dose 15 35Treatment L R L R L R Vehicle † 6.4 ± 0.5 13.9 ± 1.2  7.5 ± 0.5 12.8 ±1.1  7.0 ± 1.2 10.3 ± 0.9 5 mL/kg, i.v. (9) (9) (9) (9) (9) (9)Dihydroetorphine 6.4 ± 0.5 13.5 ± 1.0  6.9 ± 1.0 13.2 ± 1.0  7.4 ± 0.512.6 ± 1.1 (R) 0.1 μg/kg, i.v. Dihydroetorphine 6.3 ± 0.6 14.3 ± 0.6 9.4 ± 0.8 15.1 ± 0.9  9.9 ± 1.1 14.5 ± 0.7 ^(‡) (R) 0.3 μg/kg, i.v.Dihydroetorphine 6.4 ± 0.4 14.1 ± 0.9  9.6 ± 1.2 15.9 ± 1.2  9.2 ± 1.213.1 ± 1.2 (R) 0.5 μg/kg, i.v. Morphine 6.4 ± 0.4 15.4 ± 1.0 12.0 ± 1.5^($) 17.5 ± 0.5 ^($$$) 16.4 ± 0.9 *** 16.8 ± 0.7 *** 5 mg/kg, i.v.Withdrawal Latency (s) at Time (min) Post-Dose Phase 1 60 130 TreatmentL R L R Vehicle †  7.6 ± 0.9 12.4 ± 1.4  7.4 ± 0.9 14.1 ± 1.3 5 mL/kg,i.v. (9) (9) (9) (9) Dihydroetorphine  7.7 ± 1.0 12.9 ± 1.2  8.1 ± 0.614.3 ± 0.8 (R) 0.1 μg/kg, i.v. Dihydroetorphine  8.3 ± 0.8 14.6 ± 0.9 7.7 ± 0.8 14.2 ± 0.9 (R) 0.3 μg/kg, i.v. Dihydroetorphine  8.4 ± 0.613.3 ± 1.2  8.0 ± 1.1 10.9 ± 1.0 (R) 0.5 μg/kg, i.v. Morphine 12.8 ± 1.3** 16.3 ± 1.1 * 10.6 ± 0.9 * 13.8 ± 1.1 5 mg/kg, i.v. Data are expressedas Mean ± SEM. n = 10 animals per group, unless stated in parenthesis. †Vehicle = citrate buffer (citric acid monohydrate:sodium citrate:sodiumchloride:water for injection, in the ratio, 0.03:0.10:0.86:99.01(g:g:g:mL). ^($) P < 0.05 when compared to vehicle group data(Mann-Whitney U-test). ^($$$) P < 0.001 when compared to vehicle groupdata (Mann-Whitney U-test). * P < 0.05 when compared to vehicle groupdata (unpaired 2-tailed Student's t-test). ** P < 0.01 when compared tovehicle group data (unpaired 2-tailed Student's t-test). *** P < 0.001when compared to vehicle group data (unpaired 2-tailed Student'st-test). ^(‡) P < 0.05 when compared to vehicle group data (ANOVA andDunnett's t-test).

TABLE 19 Effects of intravenous S-DHE on the left (L) and right (R) pawwithdrawal thresholds to Von Frey monofilament challenges in rats (Phase2) (a) Raw Data Withdrawal Threshold (g) at Time (min) Post-Dose Phase 2Pre-Dose 5 25 Treatment L R L R L R Vehicle ^(†) 1.37 ± 0.31 21.03 ±1.84  6.11 ± 2.39 23.46 ± 1.06  1.66 ± 0.47 21.67 ± 1.61 5 mL/kg, i.v.Dihydroetorphine 0.94 ± 0.11 17.27 ± 2.12  8.69 ± 2.98 21.10 ± 1.46 8.28 ± 2.48 19.15 ± 1.55 (S) 3 μg/kg, i.v. Dihydroetorphine 1.19 ± 0.3120.94 ± 1.90 12.15 ± 2.99 23.46 ± 1.06  3.09 ± 1.35 22.57 ± 1.39 (S) 10μg/kg, i.v. Dihydroetorphine 0.84 ± 0.10 18.78 ± 1.89 24.56 ± 0.33 24.56± 0.33 21.92 ± 1.70 24.56 ± 0.33 (S) 30 μg/kg, i.v. Pregabalin 1.09 ±0.35 18.63 ± 2.21  2.83 ± 0.62 20.42 ± 2.14  3.27 ± 0.97 19.39 ± 1.61 30mg/kg, p.o. (a) Raw Data Withdrawal Threshold (g) at Time (min)Post-Dose Phase 2 50 120 Treatment L R L R Vehicle ^(†)  1.11 ± 0.1520.94 ± 1.90  1.35 ± 0.28 19.55 ± 1.69 5 mL/kg, i.v. Dihydroetorphine 0.99 ± 0.12 21.27 ± 1.53  1.19 ± 0.20 19.84 ± 1.94 (S) 3 μg/kg, i.v.Dihydroetorphine  1.48 ± 0.42 23.46 ± 1.06  0.83 ± 0.10 21.51 ± 1.55 (S)10 μg/kg, i.v. Dihydroetorphine  7.34 ± 2.47 23.67 ± 1.07  1.20 ± 0.3420.83 ± 1.99 (S) 30 μg/kg, i.v. Pregabalin 10.35 ± 2.51 21.34 ± 1.4913.90 ± 3.00 23.26 ± 1.04 30 mg/kg, p.o. (b) Log Data WithdrawalThreshold (Log 10 (force (g) × 10 000)) at Time (min) Post-Dose Phase 2Pre-Dose 5 25 Treatment L R L R L R Vehicle ^(†) 4.07 ± 0.07 5.30 ± 0.054.50 ± 0.16 ^($) 5.36 ± 0.02 4.11 ± 0.10 5.32 ± 0.04 5 mL/kg, i.v.Dihydroetorphine 3.95 ± 0.05 5.20 ± 0.06 4.60 ± 0.20 5.31 ± 0.03 4.69 ±0.17 5.27 ± 0.04 (S) 3 μg/kg, i.v. Dihydroetorphine 3.99 ± 0.08 5.30 ±0.05 4.90 ± 0.16 5.36 ± 0.02 4.26 ± 0.13 5.34 ± 0.03 (S) 10 μg/kg, i.v.Dihydroetorphine 3.90 ± 0.05 5.25 ± 0.05 5.39 ± 0.00 ^(###) 5.39 ± 0.005.32 ± 0.04 ^(###) 5.39 ± 0.00 (S) 30 μg/kg, i.v. Pregabalin 3.92 ± 0.095.24 ± 0.06 4.31 ± 0.13 5.28 ± 0.06 4.35 ± 0.13 5.27 ± 0.04 30 mg/kg,p.o. (b) Log Data Withdrawal Threshold (Log 10 (force (g) × 10 000)) atTime (min) Post-Dose Phase 2 50 120 Treatment L R L R Vehicle ^(†) 4.01± 0.06 5.30 ± 0.05 4.08 ± 0.06 5.28 ± 0.04 5 mL/kg, i.v.Dihydroetorphine 3.96 ± 0.06 5.32 ± 0.03 4.02 ± 0.07 5.27 ± 0.05 (S) 3μg/kg, i.v. Dihydroetorphine 4.05 ± 0.10 5.36 ± 0.02 3.89 ± 0.05 5.32 ±0.03 (S) 10 μg/kg, i.v. Dihydroetorphine 4.60 ± 0.17 5.37 ± 0.02 3.97 ±0.09 5.29 ± 0.05 (S) 30 μg/kg, i.v. Pregabalin 4.87 ± 0.14 *** 5.32 ±0.03 5.03 ± 0.11 *** 5.36 ± 0.02 30 mg/kg, p.o. Data are expressed asMean ± SEM. n = 10 animals per group. Statistical analysis onlyperformed on log data. ^(†) Vehicle = citrate buffer (citric acidmonohydrate:sodium citrate:sodium chloride:water for injection, in theratio, 0.03:0.10:0.86:99.01 (g:g:g:mL). *** P < 0.001 when compared topre-dose data (paired 2-tailed Student's t-test). ^(###) P < 0.001 whencompared to vehicle group data (Kruskal-Wallis and Dunn's test). ^($) P< 0.05 when compared to pre-dose data (paired 2-tailed Student'st-test).

TABLE 20 Effects of intravenous S-DHE on the left (L) and right (R) pawwithdrawal latency to a thermal plantar stimulus in rats (Phase 2)Withdrawal Latency (s) at Time (min) Post-Dose Phase 2 Pre-Dose 15 35Treatment L R L R L R Vehicle ^(†) 6.2 ± 0.5 14.8 ± 0.6 10.3 ± 0.8^($$)13.4 ± 0.8  8.1 ± 0.5^($) 11.7 ± 0.7^($) 5 mL/kg, i.v. Dihydroetorphine6.2 ± 0.6 12.6 ± 0.7  9.8 ± 0.9 15.0 ± 0.7 10.0 ± 0.7 12.1 ± 0.9 (S) 3μg/kg, i.v. Dihydroetorphine 6.2 ± 0.6 13.8 ± 0.8 10.4 ± 1.3 14.5 ± 0.910.8 ± 1.1 12.5 ± 0.9 (S) 10 μg/kg, i.v. Dihydroetorphine 6.2 ± 0.5 13.5± 0.9 17.6 ± 0.4 ^(##) 17.5 ± 0.4 ^(##) 10.1 ± 1.4 11.8 ± 1.1 (S) 30μg/kg, i.v. Pregabalin 6.2 ± 0.5 13.4 ± 0.9  8.3 ± 0.7 * 13.4 ± 0.6  8.6± 1.0 * 11.9 ± 0.8 30 mg/kg, p.o. Withdrawal Latency (s) at Time (min)Post-Dose Phase 2 60 130 Treatment L R L R Vehicle ^(†)  9.3 ± 0.7^($$$)12.1 ± 0.6^($)  9.8 ± 1.0^($$) 13.1 ± 1.0 5 mL/kg, i.v. Dihydroetorphine 9.1 ± 0.9 13.5 ± 0.8 10.4 ± 1.3 14.3 ± 0.8 (S) 3 μg/kg, i.v.Dihydroetorphine 11.3 ± 1.1 12.7 ± 0.6 11.3 ± 1.6 13.0 ± 0.8 (S) 10μg/kg, i.v. Dihydroetorphine  8.3 ± 1.0 14.3 ± 1.1  8.7 ± 0.9 14.2 ± 1.3(S) 30 μg/kg, i.v. Pregabalin  8.8 ± 1.0 * 12.9 ± 0.6  9.6 ± 0.8 ***13.4 ± 0.9 30 mg/kg, p.o. Data are expressed as Mean ± SEM. n = 10animals per group. ^(†) Vehicle = citrate buffer (citric acidmonohydrate:sodium citrate:sodium chloride:water for injection, in theratio, 0.03:0.10:0.86:99.01 (g:g:g:mL). ^(##) P < 0.01 when compared tovehicle group data (Kruskal-Wallis and Dunn's test). * P < 0.05 whencompared to pre-dose data (paired 2-tailed Student's t-test). *** P <0.001 when compared to pre-dose data (paired 2-tailed Student's t-test).^($)P < 0.05, ^($$)P < 0.01 and ^($$$)P < 0.001 when compared topre-dose data (paired 2-tailed Student's t-test).

TABLE 21 Effects of oral Pregabalin and intravenous morphine on the left(L) and right (R) paw withdrawal thresholds to Von Frey monofilamentchallenges in neuropathic rats (Phase 3) (a) Raw Data WithdrawalThreshold (g) at Time (min) Post-Dose Phase 3 Pre-Dose 60 120 TreatmentL R L R L R Vehicle ^(†) 1.87 ± 0.38 22.53 ± 1.97  4.95 ± 0.95 20.30 ±1.99  5.00 ± 2.34 18.36 ± 2.34 10 mL/kg, p.o. Pregabalin 2.59 ± 0.6323.25 ± 1.41 10.53 ± 3.25 23.26 ± 1.04 17.06 ± 2.88 23.02 ± 1.01 30mg/kg, p.o. Pregabalin 2.02 ± 0.35 20.61 ± 2.00 18.48 ± 3.27 22.57 ±1.39 13.69 ± 3.68 21.67 ± 1.61 50 mg/kg, p.o. Pregabalin 1.31 ± 0.1821.67 ± 1.93 15.53 ± 2.78 22.24 ± 1.82 23.29 ± 1.19 24.28 ± 0.37 100mg/kg, p.o. Morphine 1.46 ± 0.37 21.26 ± 2.11 21.32 ± 2.56 24.02 ± 0.3411.08 ± 2.85 20.04 ± 1.81 5 mg/kg, i.v. (9) (9) (9) (9) (9) (9) (a) RawData Withdrawal Threshold (g) at Time (min) Post-Dose Phase 3 180 240Treatment L R L R Vehicle ^(†)  2.57 ± 0.92 16.57 ± 1.75  1.48 ± 0.3020.54 ± 1.93 10 mL/kg, p.o. Pregabalin 13.86 ± 3.21 22.74 ± 0.97  8.02 ±2.65 23.99 ± 0.38 30 mg/kg, p.o. Pregabalin 15.20 ± 3.31 24.20 ± 0.3912.05 ± 3.41 22.74 ± 0.97 50 mg/kg, p.o. Pregabalin 19.77 ± 2.70 23.70 ±1.04 15.91 ± 2.86 23.18 ± 1.00 100 mg/kg, p.o. Morphine  3.68 ± 0.9719.57 ± 1.75  1.29 ± 0.20 18.71 ± 1.81 5 mg/kg, i.v. (9) (9) (9) (9) (b)Log Data Withdrawal Threshold (Log 10 (force (g) × 10 000)) at Time(min) Post-Dose Phase 3 Pre-Dose 60 120 Treatment L R L R L R Vehicle^(†) 4.20 ± 0.08 5.33 ± 0.05 4.57 ± 0.13 ^($$) 5.28 ± 0.05 4.43 ± 0.155.22 ± 0.07 10 mL/kg, p.o. Pregabalin 4.29 ± 0.11 5.35 ± 0.03 4.70 ±0.20 5.36 ± 0.02 5.13 ± 0.11 ^(##) 5.36 ± 0.02 30 mg/kg, p.o. Pregabalin4.25 ± 0.07 5.29 ± 0.05 5.10 ± 0.16 5.34 ± 0.03 4.85 ± 0.19 5.32 ± 0.0450 mg/kg, p.o. Pregabalin 4.07 ± 0.06 5.31 ± 0.05 5.08 ± 0.12 5.33 ±0.05 5.36 ± 0.03 ** 5.38 ± 0.01 100 mg/kg, p.o. Morphine 4.08 ± 0.095.30 ± 0.05 5.22 ± 0.15 ⁺⁺⁺ 5.38 ± 0.01 4.89 ± 0.14 ⁺⁺ 5.29 ± 0.04 5mg/kg, i.v. (9) (9) (9) (9) (9) (9) (b) Log Data Withdrawal Threshold(Log 10 (force (g) × 10 000)) at Time (min) Post-Dose Phase 3 180 240Treatment L R L R Vehicle ^(†) 4.23 ± 0.12 5.20 ± 0.05 4.11 ± 0.07 5.29± 0.05 10 mL/kg, p.o. Pregabalin 4.95 ± 0.16 ** 5.35 ± 0.02 4.68 ± 0.165.38 ± 0.01 30 mg/kg, p.o. Pregabalin 5.00 ± 0.15 ** 5.38 ± 0.01 ^(#)4.83 ± 0.18 ^(#) 5.35 ± 0.02 50 mg/kg, p.o. Pregabalin 5.23 ± 0.09 **5.37 ± 0.02 ^(##) 5.11 ± 0.10 ^(###) 5.36 ± 0.02 100 mg/kg, p.o.Morphine 4.45 ± 0.11 ⁺ 5.28 ± 0.04 4.07 ± 0.07 5.26 ± 0.04 5 mg/kg, i.v.(9) (9) (9) (9) Data are expressed as Mean ± SEM. n = 10 animals pergroup, unless stated in parenthesis. Statistical analysis only performedon log data. ^(†) Vehicle = 1% carboxymethylcellulose. ⁺ P < 0.05, ⁺⁺ P< 0.01 and ⁺⁺⁺ P < 0.001 when compared to pre-dose data (paired 2-tailedStudent's t-test). ^(#) P < 0.05, ^(##) P < 0.01 and ^(###) P < 0.001when compared to vehicle group data (Kruskal-Wallis and Dunn's test). **P < 0.01 when compared to vehicle group data (ANOVA and Dunnett's test).^($$) P < 0.01 when compared to pre-dose data (paired 2-tailed Student'st-test).

TABLE 22 Effects of oral Pregabalin and intravenous morphine on the left(L) and right (R) paw withdrawal latency to a thermal plantar stimulusin neuropathic rats (Phase 3) Withdrawal Latency (s) at Time (min)Post-Dose Phase 3 Pre-Dose 70 130 Treatment L R L R L R Vehicle ^(†) 6.5± 0.5 12.4 ± 0.8  9.2 ± 1.0 12.2 ± 0.5 9.0 ± 1.0 10.7 ± 0.7 10 mL/kg,p.o. Pregabalin 6.5 ± 0.5 13.1 ± 0.6  9.9 ± 0.8 13.4 ± 1.0 9.3 ± 0.813.7 ± 0.9 30 mg/kg, p.o. Pregabalin 6.5 ± 0.6 11.4 ± 0.8 10.6 ± 0.612.9 ± 0.9 9.7 ± 1.1 13.6 ± 1.1 50 mg/kg, p.o. Pregabalin 6.6 ± 0.7 11.6± 0.5 11.5 ± 0.8 12.6 ± 0.9 9.9 ± 0.9 13.4 ± 1.3 100 mg/kg, p.o.Morphine 6.0 ± 0.5 11.8 ± 0.6 11.8 ± 1.2 ⁺⁺ 14.0 ± 1.2 7.3 ± 0.9 13.1 ±1.0 5 mg/kg, i.v. (9) (9) (9) (9) (9) (9) Withdrawal Latency (s) at Time(min) Post-Dose Phase 3 190 250 Treatment L R L R Vehicle ^(†)  9.3 ±1.2 12.2 ± 1.0  9.5 ± 0.8 ^($$) 11.4 ± 0.6 10 mL/kg, p.o. Pregabalin 8.3 ± 1.1 12.2 ± 0.4 10.9 ± 0.8 14.0 ± 0.8 30 mg/kg, p.o. Pregabalin11.2 ± 1.1 12.7 ± 0.5  9.0 ± 0.7 11.9 ± 0.7 50 mg/kg, p.o. Pregabalin 7.6 ± 0.5 13.7 ± 0.7  9.7 ± 0.8 13.7 ± 1.1 100 mg/kg, p.o. Morphine 8.0 ± 0.8 ⁺ 14.3 ± 1.0 10.6 ± 1.4 ⁺ 14.1 ± 0.9 5 mg/kg, i.v. (9) (9)(9) (9) Data are expressed as Mean ± SEM. n = 10 animals per group,unless stated in parenthesis ^(†) Vehicle = 1% carboxymethylcellulose. ⁺P < 0.05 and ⁺⁺ P < 0.01 when compared to pre-dose data (paired 2-tailedStudent's t-test). ^($$) P < 0.01 when compared to pre-dose data (paired2-tailed Student's t-test

The invention claimed is:
 1. A process for the preparation of a compoundof formula (VI), or a salt or derivative thereof,

wherein R¹ and R² are independently C₁₋₈ straight-chain alkyl and *represents a (S) stereocentre, comprising reacting a compound of formula(IV)

wherein R¹ is as defined above, with a compound of formula R²M(X)_(p),wherein R² is C₁₋₈ straight-chain alkyl, M is metal, X is halide and pis 1 or 0, to produce: a compound of formula (V)

wherein R¹, R² and * are as defined above, and hydrolyzing said compoundof formula (V) to produce a compound of formula (VI).
 2. The process asclaimed in claim 1, wherein said compound of formula (IV) is prepared byreducing a compound of formula (III)

wherein R¹ is as defined in claim
 1. 3. The process as claimed in claim2, wherein said compound of formula (III) is prepared by reacting acompound of formula (I)

with a compound of formula (II)

wherein R¹ is C₁₋₈ straight-chain alkyl.
 4. A process for thepreparation of a compound of formula (VI), or a salt or derivativethereof,

wherein R¹ and R² are independently C₁₋₈ straight-chain alkyl and *represents a (S) stereocentre, comprising: reacting a compound offormula (I)

with a compound of formula (II)

wherein R¹ is C₁₋₈ straight-chain alkyl, to give a compound of formula(III)

reducing said compound of formula (III) to produce a compound of formula(IV)

reacting said compound of formula (IV) with a compound of formulaR²M(X)_(p), wherein R² is C₁₋₈ straight-chain alkyl, M is metal, X ishalide and p is 1 or 0, to give a compound of formula (V)

and hydrolysing said compound of formula (V) to give a compound offormula (VI).
 5. The process as claimed in claim 1 or claim 4, furthercomprising the step of crystallising said compound of formula (VI). 6.The process as claimed in claim 1 or claim 4, wherein the compound offormula (V) is:


7. The process as claimed in claim 1 or claim 4, wherein the compound offormula (VI) is:


8. A compound of formula (VI), or a salt or derivative thereof,

wherein R¹ and R² are independently C₁₋₈ straight-chain alkyl and *represents a (S) stereocentre.
 9. The compound as claimed in claim 8,having the formula (VIa), or a salt or derivative thereof


10. A compound of formula (V), or a salt or derivative thereof,

wherein R¹ and R² are independently C₁₋₈ straight-chain alkyl and *represents a (S) stereocentre.
 11. The compound as claimed in claim 10,having the formula (Va), or a salt or derivative thereof


12. The process as claimed in claim 1 or claim 4, wherein R¹ is C₃₋₅alkyl.
 13. The process as claimed in claim 12, wherein R¹ is propyl. 14.The process as claimed in claim 13, wherein R¹ is n-propyl.
 15. Theprocess as claimed in claim 1 or claim 4, wherein R² is C₁₋₂ alkyl. 16.The process as claimed in claim 15, wherein R² is methyl.
 17. Theprocess as claimed in claim 1 or claim 4, wherein the compound offormula (V) is produced in a diastereomeric excess of at least 90%. 18.The compound as claimed in claim 8 in free base form.
 19. The compoundas claimed in claim 9 in free base form.
 20. The compound as claimed inclaim 10 in free base form.
 21. The compound as claimed in claim 8 as apharmaceutically acceptable salt.
 22. The compound as claimed in claim21 as a hydrochloride salt.
 23. The compound as claimed in claim 9 as apharmaceutically acceptable salt.
 24. The compound as claimed in claim23 as a hydrochloride salt.
 25. The compound as claimed in claim 10 as apharmaceutically acceptable salt.
 26. The compound as claimed in claim25 as a hydrochloride salt.
 27. A transdermal dosage form, comprising acompound as claimed in any one of claims 8 to
 10. 28. The transdermaldosage form as claimed in claim 27 in the form of a patch.
 29. Thecompound of claim 11 in free base form.
 30. The compound as claimed inclaim 11 as a pharmaceutically acceptable salt.
 31. The compound asclaimed in claim 30 as a hydrochloride salt.
 32. The process as claimedin claim 1 or claim 4, wherein M is magnesium or lithium.
 33. Theprocess as claimed in claim 1 or claim 4, wherein R²M(X)_(p) is R²MgCl,R²MgBr, R²MgI, or R²MgLi.
 34. A pharmaceutical composition comprising acompound as claimed in any one of claims 8 to 11, claims 18 to 26, orclaims 29 to
 31. 35. A method of treating, a subject in need of painrelief, comprising administering to said subject a therapeuticallyeffective amount of a compound as claimed in any one of claims 8 to 11,claims 18 to 26, or claims 29 to
 31. 36. The pharmaceutical compositionas claimed in claim 34, wherein said composition is in a dosage formsuitable for transdermal administration.